Response to the External Peer Review of

“Technical Review of Hexabromocyclododecane”

January 11, 2016

United States Environmental Protection Agency

Office of Environmental Information

Office of Information Access and Analysis
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Table of Contents

Background...................................................................................................4

Response to External Peer Review Section I: Chemistry and Environmental Fate.............4

I. Charge to Reviewers........................................................................................4

II. Response to Chemistry and Environmental Fate Assessment Charge.................................4

A. General Impressions..............................................................................4

B. Peer Reviewer Comments by Charge Question... .........................................5

1. Are there any additional chemical properties of HBCD that should be 

included in the technical review?............................................................................5

2. Do the data support a conclusion that the half-life of HBCD is at least 

60 days in water, sediment, soil?.............................................................................8

3. Does the technical review accurately address the fate of HBCD in the 

environment, including transformations resulting in the formation of toxic 

chemicals?..............................................................................................................9

4. Please provide any additional comments or suggestions you may have 

that may improve this section of the technical review. ...............................13

Response to External Peer Review Section II: Human Health Effects...........................17

I. Charge to Reviewers.......................................................................................17

II. Response to Human Health Hazard Assessment Charge.............................................17

A. General Impressions.............................................................................17

B. Peer Reviewer Comments by Charge Question..............................................17

1. Does the summary statement and review of the literature present a clear 

rationale and accurately reflect the conclusions regarding the human 

health hazards of HBCD?......................................................................................17

2a. Please comment on the non-cancer endpoints. Has the scientific evidence for 

non-cancer endpoints been accurately and clearly represented and evaluated? Do 

you agree or disagree with the conclusions?..........................................................24

2b. Please comment on the adequacy of the conclusions in the HBCD hazard 

assessment with regard to toxicity in the liver and thyroid...................................28

2c. Please comment on the adequacy of the conclusions in the HBCD hazard 

assessment with regard to neurotoxicity. ..............................................31

2d. Please comment on the adequacy of the conclusions in the HBCD hazard 

assessment with regard to other developmental and reproductive effects. ........32

3. Do you agree or disagree with the selection of the LOAELs/NOAELs (or 

BMDLs/BMDs) for all endpoints?.....................................................................35

4. Are you aware of any additional key published studies or publicly available 

scientific reports that are missing from the draft document that might be useful for 

discussion of the hazards? If so, please provide a copy of the study or a complete 

citation. .....................................................................................39

5. Please provide any additional comments or suggestions you may have that may 

improve this section of the document. ..................................................42
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Response to External Peer Review Section III: Ecological Hazard Assessment................45

I. Charge to Reviewers.......................................................................................45

II. Response to Ecological Hazard Assessment Charge................................................45

A. General Impressions.............................................................................45

B. Peer Reviewer Comments by Charge Question..............................................45

1. Does the summary statement and review of the literature present a clear 

rationale and accurately reflect the conclusions regarding the ecological hazards 

of HBCD?..............................................................................................................45

2. Please comment on the characterization of HBCD’s potential for 

bioaccumulation. Has the scientific evidence been accurately and clearly 

represented and evaluated? Do you agree or disagree with the 

conclusions?...........................................................................................................53

3. Are there additional key published studies or publicly available scientific 

reports that are missing from the draft document that might be useful for 

discussion of the ecological hazards?....................................................................58

4. Please provide any additional comments or suggestions you may have that may 

improve this section of the document. ..................................................62
4

External Peer Review of EPA’s Draft Technical Assessment of 

Hexabromocyclododecane (HBCD)

BACKGROUND

Hexabromocyclododecane (HBCD) is not currently listed on the Toxics Release Inventory (TRI); 

therefore, companies are not required to report releases of this substance. The EPA Office of 

Chemical Safety and Pollution Prevention included adding HBCD to the TRI in their HBCD Action 

Plan released in 2010 (EPA, 2010). The EPA Office of Environmental Information (OEI) has 

prepared a technical review document to assess the chemistry and environmental fate, human health 

effects, and ecological hazards of HBCD. HBCD is a brominated flame retardant consisting of a 12-

membered carbon ring with 6 bromine atoms attached at the 1, 2, 5, 6, 9, and 10 positions. HBCD is 

widely used as an additive to impart flame-retardant properties to plastics and textiles. 

The purpose of this peer review is to seek comment on the accuracy and completeness of EPA’s 

draft technical assessment of HBCD (U.S. EPA, 2013).

Response to External Peer Review Section I: Chemistry and Environmental Fate

I. Charge to Reviewers

1. Are there any additional chemical properties of HBCD that should be included in the 

technical review?

2. Do the data support a conclusion that the half-life of HBCD is at least 60 days in water, 

sediment, soil? 

3. Does the technical review accurately address the fate of HBCD in the environment, 

including transformations resulting in the formation of toxic chemicals?

4. Please provide any additional comments or suggestions you may have that may improve this 

section of the technical review. 

II. Response to Chemistry and Environmental Fate Assessment Charge 

A. General Impressions

Peer Reviewer 1 Comment: A Web of Science search for the terms hexabromocyclododecane and 

environmental or pollutant returned 240 peer reviewed citations dealing with the environmental fate 

and effects of this material. The section I reviewed included approximately 28 references, 17 of 

which were secondary references, including Federally produced data summary documents and 

extremely general references like the CRC Handbook. Given the confusion regarding melting point, 

water solubility and composition (e.g., melting points cited as varying by 40oC) this was a 

disappointingly shallow review. For a point of reference, had a student turned in a review of 

physical/chemical properties like this as an assignment for my Environmental Chemistry class I 

would have returned the paper ungraded with a “do-over” instruction or it would have been marked 

“incomplete.” This is particularly disappointing given the economic and health-related impacts of 

this chemical.

EPA Response: A great deal of research has been conducted on HBCD so one would expect that a 

general search would find many citations. However, the fact that there are many citations and EPA 
5

cited 28 does not mean that EPA missed relevant information. Some of the review articles cited by 

EPA covered many other publications and there are duplications of information in many of the 

available sources. Nevertheless, EPA has added numerous primary articles to this section such that 

there are now over 70 references cited. With regard to the physical properties issue, HBCD is a 

mixture of diastereomers which have significant differences in physical properties, for example the 

various HBCD mixtures have large differences in melting points. It should also be noted that at 190 

oC the diastereomers begin to thermally re-arrange, and decomposition was reported as the 

temperature in which 5% of the original material was lost. The melting point takes a wide range of 

values because different mixtures have different compositions of diastereomers; thus, there is no 

single well-defined melting point for HBCD. Where available, EPA added additional information 

on the physical properties of the individual diastereomers which provides a better understanding of 

the observed differences in the physical properties of HBCD mixtures.

Peer Reviewer 2 Comment: While my specific charge was to focus my review on pages 4 to 19, I 

found material relevant to the charge in other sections of the Assessment, and I therefore have 

included consideration of this material when preparing my review.

EPA Response: No action is required based on this comment. 

B. Peer Reviewer Comments by Charge Question

1. Are there any additional chemical properties of HBCD that should be included in the 

technical review?

Peer Reviewer 1 Comment: Yes.

Physical and chemical property data should be based on original literature only. The current 

document cites compilations as having the same value as primary sources; this is not acceptable.

When measured, properties should have error reported. The current format reports data such as 

melting point in a tabular format with multiple listings rather than as mean, median or any value 

with an error range. 

When estimated, properties derived from models (e.g., EPI Suite) should have error of the 

model reported. Various modeled properties are cited with the same weight given experimentally 

derived data, with no reference to the accuracy or precision of the given model. 

In summary, all properties should be given with associated error ranges based on their method of 

determination.

EPA Response: Where available/feasible EPA has added primary sources and error ranges to 

provide a more robust discussion of the physical/chemical properties. Many of the 

physical/chemical properties tests were conducted by Wildlife International for the Brominated 

Flame Retardant Industry Panel’s High Production Volume submission to EPA. EPA was able to 

access several of these studies to cite as primary sources. Another primary study of interest that 

was added was Peled et al. 1995 (see below), a primary source for boiling point/decomposition data

that discusses interconversion of the diastereoisomers. 
6

Peled M, Scharia R and Sondack D. Thermal rearrangement of hexabromocyclododecane (HBCD). 

In Industrial Chemistry Library. Advances in organobromine chemistry II. Edited by Desmurs J-R, 

Gérard B and Goldstein MJ. 1995 

The physical and chemical properties table has been revised as follows:

Table 2-1. Physical and chemical properties of HBCD

Property Valuea Reference

Molecular 

formula

C12H18Br6

Molecular 

weight

641.695 Lide, 2008

Melting point 172-184 °C (crude product)

201-205 °C (highest-melting version)

179-181 °C (α-HBCD)

170-172 °C (β-HBCD)

207-209 °C (γ-HBCD) 

190±20 °C (average)

Smith et al., 2005

Boiling point Not applicable; thermally induced 

isomerization occurs at 160 to 220 °C and 

decomposition occurs at >220 °C 

Peled et al., 1995; Heeb et al., 2008

Vapor pressure 4.70×10-7 mm Hg (6.27×10-5 Pa) at 21 °C 

(technical product)

Stenzel and Nixon, 1997

Water solubility 8.6 μg/L at 25 °C (14C-labeled technical 

product)

Great Lakes Chemical Corp., 1990 as 

cited in HSDB, 2011

3.4±2.3 μg/L at 25 °C (based on γ-HBCD in 

technical product reported with coefficient of 

variation)

Stenzel and Markley, 1997 as cited in 

U.S. EPA, 2005b, ECB, 2008 and 

Marvin et al., 2011

48.8±1.9 μg/L (α-HBCD)

14.7±0.5 μg/L (β-HBCD)

2.1±0.2 μg/L (γ-HBCD)

ƩHBCD 65.6 μg/L

MacGregor and Nixon, 2004 as cited in 

U.S. EPA, 2005b, ECB, 2008 and 

Marvin et al., 2011

Log Kow 5.625 at 25±0.05°C (technical product) Hayward et al., 2006; as cited in U.S. 

EPA, 2005b, ECB, 2008 and Marvin et 

al., 2011 

5.07±0.09 (α-HBCD)

5.12±0.09 (β-HBCD)

5.47±0.10 (γ-HBCD) (estimated)

MacGregor and Nixon, 1997 as cited in 

U.S. EPA, 2005b ECB, 2008, and 

Marvin et al., 2011

Koc 7.6×104(estimated) U.S. EPA, 2011b (EPISuiteTM v4.10)

Henry’s Law 

constant

4.6×10-5atm-m3/mol (calculated) 

Derived from a vapor pressure of 

4.70×10-7 mm Hg and water solubility of 

8.6 μg/L

VP: Stenzel and Nixon, 1997 

WS: Great Lakes Chemical Corp., 

1990 as cited in HSDB, 2011

Density 2.24 g/cm3

2.38 g/cm3

(technical product)

Great Lakes Chemical Corporation, 

1994 and Albemarle Corporation, 1994 

as cited in ECB, 2008

Flash point Not applicable

aValues are experimental unless otherwise indicated.
7

Peer Reviewer 2 Comment: While the assessment covers the chemical properties necessary to 

describe the environmental fate of HBCD in general, more detailed evidence of how such properties 

vary between the different diastereomers is needed. This is particularly important, given the 

observed shift from predominantly -HBCD on the technical mixture and treated products to 

predominantly (almost exclusively -HBCD in biota including humans). Data is also required on 

the interconversion between diastereomers which has been observed to occur via both biological, 

thermal, and photolytic mechanisms. There is also no mention made of the observed enrichment of 

the (-)--HBCD enantiomer in humans. While we do not yet understand what the implications of 

this might be, it is surely still worth noting.

EPA Response: Where available, EPA has included additional details regarding the environmental 

fate of specific isomers. As shown below, EPA also added a new section “2.6.1.1. Diastereomers 

and isomerization” which includes a discussion of the study by Peled et al. (1995) that describes the 

thermal interconversion of HBCD isomers. 

Peled M, Scharia R and Sondack D. Thermal rearrangement of hexabromocyclododecane (HBCD). 

In Industrial Chemistry Library. Advances in organobromine chemistry II. Edited by Desmurs J-R, 

Gérard B and Goldstein MJ. 1995

“2.6.1.1. Diastereomers and isomerization. As reported in Section 2.4, γ-HBCD is the dominant 

stereoisomer present in most commercial HBCD formulations (>75%) (Becher, 2005). Similar 

proportionality is observed with HBCD stereoisomers detected in abiotic environmental media 

(Covaci et al., 2006; Marvin et al., 2011). However, in living organisms, α-HBCD is the 

predominant stereoisomer identified, accounting for 70–90% of all HBCD detected (Covaci et al., 

2006; Harrad et al., 2009b; de Wit et al., 2010; Marvin et al., 2011). For example, the majority of 

the HBCD detected in human milk is α-HBCD. In milk samples from Birmingham, United 

Kingdom, 62–95% of the total HBCD was α-HBCD (Abdallah and Harrad, 2011). In milk samples 

from Boston, Massachusetts, HBCD was detected in all 43 samples and α-HBCD constituted 40–

84% of the total HBCD (Carignan et al., 2012).

The reasons for the difference in HBCD composition found in living organisms compared to that 

found in the abiotic environment have not been determined. Possible explanations include different 

rates of bioconcentration for the α , β-, and γ- stereoisomers and diastereomer interconversion and 

metabolism within organisms (Covaci et al., 2006; Law et al., 2006; ECB, 2008; Marvin et al., 

2011; NICNAS, 2012). In the Western Scheldt (Netherlands) estuarine food web, γ-HBCD 

concentrations decreased and α-HBCD concentrations increased with an increase in trophic level 

(Brandsma et al., 2015). This finding suggests differential bioaccumulation/ biomagnification of 

diastereomers. A possible example of diastereomer interconversion is detection of α-HBCD in 

rainbow trout fed for 168 days on food containing only β- or γ-HBCD in statistically significant 

amounts (Law et al., 2006). It is not yet clear to what extent the levels of specific HBCD 

diastereomers found in the environment and biota arise from interconversion processes. The rates 

and mechanisms of conversion between the HBCD diastereomers and the importance of these 

processes to environmental fate is an active area of current research.”

Peer Reviewer 3 Comment: The technical review provides a comprehensive assessment of the 

primary chemical/ physical properties required to assess key physical properties, including 

persistence. I would also recommend adding a table that includes key characteristics directly related 
8

to chemical properties, including half-lives in different abiotic matrices, i.e., air, water, soil, 

sediment (both aerobic and anaerobic).

EPA Response: EPA added “Table 2-2. Environmental Degradation of HBCD” that provides a 

summary of the environmental fate and persistence characteristics of HBCD. The table includes 

data on photodegradation in air, hydrolysis in water, and aerobic/anaerobic degradation in sediment 

and soil. The primary citations were:

Davis, JW; Gonsior, SJ; Marty, GT; et al. (2005) The transformation of hexabromocyclododecane 

in aerobic and anaerobic soils and aquatic sediments. Water Res. 39:1075–1084.

Davis, JW; Gonsior, SJ; Markham, DA; et al. (2006) Biodegradation and product identification of 

[14C]hexabromocyclododecane in wastewater sludge and freshwater aquatic sediment. Environ.

Sci. Technol. 40:5395–5401.

2. Do the data support a conclusion that the half-life of HBCD is at least 60 days in water, 

sediment, soil?

Peer Reviewer 1 Comment: They do not. The data presented are not presented with sufficient 

coverage of peer-reviewed source literature for reliable estimates. For example, in the document a 

collection of melting points is presented that cover the range 172oC - 209oC (Table 2-1). Yet the 

EPI Suite calculation uses the temperature 190oC with no justification presented for the selection. In 

fact the same table reports decomposition at temperatures in excess of 190oC. Similarly there is no 

universal application of significant figures for the constants. 

EPA Response: EPA responded to the issues concerning chemical properties in a previous 

comment response. The commenter did not respond directly to the question about the half-life of 

HBCD in water, sediment, or soil. EPA added a discussion clarifying the issue of thermal 

degradation, as discussed in the primary source Peled et al. 1995. At 190 oC, the diastereoisomers 

began to thermally re-arrange, and decomposition was reported as the temperature in which 5% of 

the original material was lost. The melting point takes a wide range of values because different 

mixtures have different compositions of diastereoisomers; thus, there is no single well-defined 

melting point for HBCD. The melting point range and error range are not likely to answer whether 

the half-life of HBCD is 60 days or greater in water, soil and sediment. The melting point value 

chosen for EPIWIN modeling has no effect on any of the other properties calculated in the program 

and used in the assessment except the distribution of the substance in the 4 environmental 

compartments in the fugacity calculation, and this effect is very minor. EPA has added water 

solubility, vapor pressure and octanol/water partition coefficient tests conducted by WildLife 

International and enhanced the discussion of physical/chemical/properties as done in the reference 

ECB 2008. The use of significant figures in the document was also reviewed.

ECB (European Chemicals Bureau). (2008) Risk assessment hexabromocyclododecane. CAS-No.: 

25637-99-4. EINECS-No.: 247-148-4. Final report. European Union. Available online at 

http://echa.europa.eu/documents/10162/661bff17-dc0a-4475-9758-40bdd6198f82 (accessed May 

20, 2015).

Peer Reviewer 1 Comment: Also data presented on the bottom of page 5 seems to directly 

contradict the statement, showing much longer half-lives.
9

EPA Response: The commenter appears to refer to the simulation tests in which 6-60% 

degradation was observed in >100 days depending upon oxygen conditions. The question EPA 

posed was whether the half-life of HBCD is at least 60 days in water, sediment, or soil. Much 

longer half-lives would support the conclusion that the half-life is “at least” 60 days. EPA clarified 

any contradictory statements with respect to persistence of HBCD and added data with respect to 

degradation rates for the individual isomers where available.

Peer Reviewer 2 Comment: The assessment provides adequate evidence to support this 

conclusion – although I would stress the need for further research into this topic in order to provide 

a more robust database on which this conclusion may be based. Moreover, as indicated above, 

greater understanding of the interconversion between different diastereomers is required if full 

understanding/appreciation of the persistence of HBCDs is to be obtained.

EPA Response: EPA included a new table “Environmental Degradation of HBCD” that provides 

additional data on the half-lives of HBCD under various environmental conditions. Where 

available, EPA incorporated additional data on isomer specific degradation kinetics and the 

interconversion of the three main diastereoisomers into the assessment. As previously noted, EPA 

added a section “2.6.1.1. Diastereomers and isomerization” that provides information on 

interconversion of the diastereomers.

Peer Reviewer 3 Comment: Overall, the data support a conclusion that the half-life is at least 60 

days in water, sediment and soil. To my knowledge, the current understanding of HBCD is 

unequivocal in that there is overwhelming evidence that HBCD is persistent in water and soil. 

However, there has been debate as to the persistence of HBCD in sediments. Studies of the 

persistence of HBCD in sediments have determined a wide range of half-lives dependent on 

concentration. Studies cited in the current document on page 12, i.e., Davis et al. (2006); ECB, 

2008; USEPA, 2005, used very high HBCD concentrations, which reportedly skew half-life 

calculations to the high side, i.e., they are artificially high. However, the sediment half-life values 

cited in the report are within the generally accepted range.

EPA Response: EPA added data by Davis et al. (Water Res. 39: 1075-1084 (2005)), which 

summarize degradation of HBCD using OECD 307 and 308 (aerobic and anaerobic degradation in 

soils and aquatic sediment). EPA also qualified the language concerning high spiking rates that 

may be toxic to the microorganisms and lead to longer half-lives:

“Although very high spiking rates can be toxic to microorganisms in biodegradation studies and 

lead to unrealistically long estimated half-lives, the results of this study did not suggest toxicity to 

microorganisms. Tests with viable microbes demonstrated increased HBCD degradation compared 

to the biologically inhibited control studies (described in Section 2.6.2)”

3. Does the technical review accurately address the fate of HBCD in the environment, 

including transformations resulting in the formation of toxic chemicals?

Peer Reviewer 1 Comment: It does not. The general problem here is that the review uses 

thermodynamic concepts to make kinetically based decisions. For example, on page 5 there is a 

comment: “HBCD is expected to volatilize slowly from moist soils and water surfaces based on its 

Henry’s Law constant of 4.6x10-5atm-m

3

/mol.” This statement is factually incorrect, in that the 
10

Henry’s Law constant is an estimator of the equilibrium position of a system, not a rate. The correct 

statement would read: The equilibrium position of HBCD will favor partitioning into moist soils or 

waters as indicated by the Henry’s Law constant. This is not a trivial problem, because it calls into 

question the assumptions made by they modelers. It seems to be an error throughout the manuscript.

EPA Response: The Henry’s Law constant can be used to estimate the overall liquid phase mass 

transfer coefficient of a substance from a water body. Using the mass transfer coefficient and the 

properties of the water body, a volatilization half-life can be calculated. This is fairly well 

understood. The text was edited to include some additional information about the model 

assumptions and the importance that adsorption to suspended solids and sediment has in reducing 

the rate of volatilization.

“HBCD is expected to volatilize slowly from moist soils and water surfaces based on its Henry’s 

Law constant of 4.6×10 5 atm-m3/mol (Great Lakes Chemical Corp., 1990 as cited in HSDB, 2011; 

Lyman et al., 1990; Stenzel and Nixon, 1997). Estimated volatilization half-lives are 34.8 hours 

from a model river and 24.7 days from a model lake, assuming no adsorption to suspended solids or 

sediment (U.S. EPA, 2011b). Volatilization is expected to be attenuated by adsorption. Therefore, 

the rate of HBCD volatilization from water will decrease with increasing amount of suspended 

particulates. The half-life for the volatilization of HBCD from a model pond has been estimated to 

be months to years if adsorption is considered (U.S. EPA, 2005a; HSDB, 2011).

Peer Reviewer 1 Comment: The section on reduction is troublesome. Is there no nucleophile that 

could have given these products? The proposed mechanism seems very clean.

EPA Response: The debromination scheme that was presented was proposed by the authors of the 

article (Davis et al., 2006, ECB 2008) and is consistent with other degradation mechanisms 

proposed for HBCD. No changes were made based on this comment.

Peer Reviewer 2 Comment: While many aspects are covered, there appears no coverage of the 

aqueous phase partitioning of HBCDs. Some data reporting the relative partitioning of HBCDs 

between the operationally-defined particulate and freely dissolved phases of lake water are not 

mentioned (Harrad et al., 2009a). Moreover, while some data on BCFs, BMFs, and BAFs are 

reported, a study reporting field BAFs for freshwater lake fish is not considered (Harrad et al., 

2009a).

EPA Response: Data from Harrad et al. 2009 were added to the assessment, for example:

“In an aqueous phase partitioning study, 47±4.7% HBCD (sum of diastereomers) detected from 

freshwater lakes was in the freely-dissolved phase, defined as capable of passing through a 1 μm 

pore size glass fiber filter. The remainder, 53±5.4% HBCD, was associated with the particulate 

phase (Harrad et al., 2009b). The measured vapor pressure suggests that HBCD will not volatilize 

from dry soil surfaces (Stenzel and Nixon, 1997; HSDB, 2011).”

While not the subject of this section of the document, bioaccumulation data have been updated and 

expanded in the Ecological Hazard Assessment section of the document.

Peer Reviewer 2 Comment: The coverage of transformation products is extremely limited and 

omits completely the available literature on the environmental presence of dehydrobromination (as 

opposed to debromination) products (specifically pentabromocyclododecenes (PBCDs) and 

tetrabromocyclododecadienes (TBCDs)) in a variety of environmental matrices, including lacustrine 
11

sediment, indoor dust, and human milk (Abdallah and Harrad, 2011; Carignan et al., 2012; Harrad 

et al., 2009a,b). It should also be noted that while lab and in vitro biological metabolism studies 

show that PBCDs and TBCDs are formed both photolytically (Harrad et al., 2009b; Kajiwara et al., 

2013) and metabolically (Abdallah et al., 2014); it is not yet clear to what extent the levels found in 

the environment arise from such processes, as opposed to their demonstrated presence as impurities 

in the technical HBCD product, as well as thermal production during the process of treating 

consumer articles with HBCD (Barontini et al., 2001). While no empirical assessment of the

toxicity of these products has yet been conducted, one computational study based on a QSAR 

approach revealed higher binding affinities of PBCDs to the human transthyretin receptor (hTTR) 

than the parent HBCDs. Interestingly, two transconfigured PBCDs showed higher binding affinities 

to hTTR than T4 itself (Weber et al., 2009). Moreover, there is literature that shows that fish are 

capable of producing the -HBCD meso form via bioisomerisation (Abdallah et al., 2014; Harrad et 

al., 2009a). Furthermore, the in vitro metabolism study of Abdallah et al. (2014) identified both 

mono- and dhydroxy metabolites of HBCD as well as mono-hydroxy metabolites of PBCDs and 

TBCDs. These findings are consistent with those of earlier studies by Brandsma et al. (2009), 

Esslinger et al. (2011), and Hakk et al. (2012).

EPA Response: Not all transformation products are intended to be covered under the section on 

“Toxic Transformation Products” however, the section has been updated and revised as follows:

“2.6.5. Toxic Transformation Products. Studies evaluating HBCD degradation products must take 

into account impurities formed during the synthesis of technical HBCD that are also transformation 

products of HBCD. For example, some technical-HBCD samples may contain PBCD-OHs and 

isobutoxypentabromocyclododecanes (iBPBCDs) as synthetic byproducts (Heeb et al., 2010, 2012). 

However, PBCD-OHs are also potential environmental transformation products and metabolites of 

biological HBCD transformations. Evaluation of the starting material is required to adequately 

identify the source of HBCD degradation products.

During a study of 14C radiolabeled HBCD degradation in a digester sludge experiment, 

HBCD transformation products from reductive dehalogenation included TBCD, DBCD, and 1,5,9 

cyclododecatriene (Davis et al., 2006; ECB, 2008). During a thermal degradation study using a 

heating rate of 10 °C/minute that reached temperatures of 500 °C in 100% nitrogen gas or air (21 

mol % oxygen), hydrogen bromide was the major degradation product. Other high-molecular 

weight brominated and non-brominated organic compounds, such as PBCDs, TBCD, naphthalene, 

and biphenyl were detected under experimental conditions using Fourier transform infrared 

spectroscopy (FTIR) and gas-chromatographic (GC)/MS analysis (Barontini et al., 2001). 

Debromination and hydroxylation are in vivo metabolic pathways of HBCD. In rats dosed 

orally with 30 or 100 mg HBCD/kg body weight/day for 30 days, debrominated structures (PBCDs, 

TBCD) and corresponding mono- and di-hydroxy structures (HBCD-OH, PBCD-OH, TBCD-OH, 

di-OH-HBCD, di-OH-PBCD, and di-OH-TBCD) were identified in adipose, liver, lung, and muscle 

tissues (Brandsma et al., 2009). C57BL/6 mice dosed once with 3 mg/kg body weight α 

[14C]HBCD orally were found to have detectable HBCD-OH (in liver, brain, and adipose tissues), 

PBCD-OH (in liver and adipose tissues), tri- and tetrabromohexene glutathione, 

hexabromododecanedioic acid, tribromononenoic acid, and methylmercapturate of TBCDs (in 

urine) and PBCD-OH, di-OH-PBCDs, and methylmercapturate of tribromononeneoic acid (in feces) 

(Hakk et al., 2012). 
12

In vitro metabolism studies using rat liver S9 fractions identified PBCD, TBCD, HBCD OH, 

di-OH-HBCD, PBCD-OHs, and TBCD-OHs (Abdallah et al., 2014). These metabolites were also 

identified after treatment with trout liver S9 fractions in this study. The estimated rate of 

-HBCD (half-lives of 6.43 and 38.10 minutes) and slowest for α

HBCD (half-lives of 17.14 and 134.91 minutes) in both the rat and trout liver S9 fractions, 

respectively. 

Esslinger et al. (2011) used rat liver microsomes to evaluate stereospecificity of HBCD 

metabolism. Estimated metabolic half-lives reported for (˗)-β, (+)-β, (˗)-γ, (+)-α, (˗)-α, and (+)-γ 

HBCD were 6.32, 6.35, 11.6, 14.1, 24.4, and 32.3 minutes, respectively. TBCD and PBCD were not 

detected in this experiment, but mono- and di-hydroxylated α- and γ-HBCD metabolites were 

found. In general, γ-HBCD is the dominant stereoisomer in commercial formulations and α HBCD 

is dominant in biota and higher trophic level animals (Covaci et al., 2006; Marvin et al., 2011; 

NICNAS, 2012). Although the reasons for the difference in HBCD composition found in the 

environment compared to that found in living organisms have not been determined, 

bioisomerisation of α-HBCD from γ-HBCD, preferential α-HBCD uptake, and the higher water 

solubility of α-HBCD have been suggested as possible sources of enrichment (Covaci et al., 2006; 

Law et al., 2006; ECB, 2008; Marvin et al., 2011; NICNAS, 2012).”

Peer Reviewer 3 Comment: The technical review accurately addressed the fate of HBCD in the 

environment, including transformations resulting in the formation of toxic chemicals. In the case of 

HBCD, these compounds represent degradation products of the parent compound as a result of 

abiotic/biotic processes. However, the technical review fails to identify interconversion of the 

HBCD diastereomers themselves as “transformation” processes. These interconversion processes 

are particularly relevant to environmental fate as conversion of gamma and/or beta HBCD to alpha 

HBCD can influence processes including bioaccumulation, and correspondingly biotic degradation. 

These processes can be both physical and biological in nature. Thermal processes occurring above 

approximately 180 °C result in conversion of gamma HBCD to alpha HBCD. In addition, HBCD 

has been demonstrated to undergo “biotransformation” or “bioisomerization” in fish resulting in 

interconversion of the isomers.

EPA Response: A new section on diastereomers and isomerization was added to the document as 

follows:

“2.6.1.1. Diastereomers and isomerization. As reported in Section 2.4, γ-HBCD is the dominant 

stereoisomer present in most commercial HBCD formulations (>75%) (Becher, 2005). Similar 

proportionality is observed with HBCD stereoisomers detected in abiotic environmental media 

(Covaci et al., 2006; Marvin et al., 2011). However, in living organisms, α-HBCD is the 

predominant stereoisomer identified, accounting for 70–90% of all HBCD detected (Covaci et al., 

2006; Harrad et al., 2009b; de Wit et al., 2010; Marvin et al., 2011). For example, the majority of 

the HBCD detected in human milk is α-HBCD. In milk samples from Birmingham, United 

Kingdom, 62–95% of the total HBCD was α-HBCD (Abdallah and Harrad, 2011). In milk samples 

from Boston, Massachusetts, HBCD was detected in all 43 samples and α-HBCD constituted 40–

84% of the total HBCD (Carignan et al., 2012).
13

The reasons for the difference in HBCD composition found in living organisms compared to that 

found in the abiotic environment have not been determined. Possible explanations include different 

rates of bioconcentration for the α , β-, and γ- stereoisomers and diastereomer interconversion and 

metabolism within organisms (Covaci et al., 2006; Law et al., 2006; ECB, 2008; Marvin et al., 

2011; NICNAS, 2012). In the Western Scheldt (Netherlands) estuarine food web, γ-HBCD 

concentrations decreased and α-HBCD concentrations increased with an increase in trophic level 

(Brandsma et al., 2015). This finding suggests differential bioaccumulation/ biomagnification of 

diastereomers. A possible example of diastereomer interconversion is detection of α-HBCD in 

rainbow trout fed for 168 days on food containing only β- or γ-HBCD in statistically significant 

amounts (Law et al., 2006). It is not yet clear to what extent the levels of specific HBCD 

diastereomers found in the environment and biota arise from interconversion processes. The rates 

and mechanisms of conversion between the HBCD diastereomers and the importance of these 

processes to environmental fate is an active area of current research.”

EPA also included additional data on this issue as noted in the summary section:

“Interconversion among HBCD diastereomers has been observed by biological, thermal, and 

photolytic mechanisms (Peled et al., 1995; Law et al., 2006; Harrad et al., 2009a). Results from 

studies determining physical properties of HBCD, such as water solubility and melting point, 

demonstrate the unique properties of the individual HBCD diastereomers.”

4. Please provide any additional comments or suggestions you may have that may improve 

this section of the technical review.

Peer Reviewer 1 Comment: Physical and chemical property data should be based on original 

literature only. 

EPA Response: Where possible, physical and chemical property data were cited from the original 

literature. For example, the citations below were added to Table 2-1. Physical and chemical 

properties of HBCD:

Heeb, NV; Schweizer, WB; Mattrel, P; et al. (2008) Regio- and stereoselective isomerization of 

hexabromocyclododecanes (HBCDs): Kinetics and mechanism of γ- to α-HBCD isomerization. 

Chemosphere 73:1201–1210.

MacGregor, JA; Nixon, WB. (2004) Determination of water solubility of Hexabromocyclododecane 

(HBCD) using a generator column method. Project No. 439C-138, Wildlife International Ltd. 

Easton, MD.

Peled, M; Scharia, R; Sondack, D. (1995) Thermal rearrangement of hexabromocyclododecane 

(HBCD). In: Desmurs, JR; Gérard, B; Goldstein, MJ, eds. Industrial Chemistry Library. Advances 

in organobromine chemistry II. 
14

Stenzel, J; Markley, B. (1997) Final Report. Hexabromocyclododecane (HBCD): Determination of 

the Water Solubility. Wildlife International Project Number: 439C-105. Wildlife International, Ltd. 

Easton, MD.

Peer Reviewer 1 Comment: When measured, properties should have error reported with their 

values. 

EPA Response: Where available, this data was added to the assessment. 

Peer Reviewer 1 Comment: When estimated, properties derived from models (e.g., EPI Suite) 

should have error of the model reported.

EPA Response: When provided by the models, error ranges for output values where added, 

however, discussion of the calibration and error of models such as the EPIWIN Suite is beyond the 

scope of this assessment.

Peer Reviewer 1 Comment: In summary, all properties should be given with associated error 

ranges based on their method of determination. 

EPA Response: As noted above, when available, error ranges were added. 

Peer Reviewer 2 Comment: It omits currently a fairly large body of work that describes the 

environmental fate and behaviour of HBCDs and which are referred to in my comments above. The 

omissions are particularly evident with respect to the degradation/metabolism of HBCD. A (notexhaustive) list of such papers is given below:

(1) M. Abdallah, C. Uchea, J. K. Chipman, S. Harrad. “Enantioselective Biotransformation of Hexabromocyclododecane by In Vitro Rat and 

Trout Hepatic Sub-cellular Fractions” Environmental Science & Technology, 48, 2732–2740 (2014).

(2) N. Kajiwara, J. Desborough, S. Harrad, H. Takigami “Photolysis of brominated flame retardants in textiles exposed to natural sunlight”, 

Environmental Science:Processes and Impacts, 15, 653-660 (2013).

(3) C. Carignan, M.A. Abdallah, N. Wu, W. Heiger-Bernays, M. McClean, S. Harrad, T. Webster. “Predictors of Tetrabromobisphenol-A (TBBPA) and Hexabromocyclododecanes (HBCD) in Milk from Boston Mothers”, Environmental Science & Technology 46, 12146-12153 (2012).

(4) M. Abdallah, S. Harrad. “Tetrabromobisphenol-A, Hexabromocyclododecane and Its Degradation Products in UK Human Milk: Relationship 

to External Exposure”, Environment International, 37, 443–448 (2011).

(5) S. Harrad, M. A. Abdallah, N. L. Rose, S. D. Turner, T. A. Davidson “Current-Use Brominated Flame Retardants in Water, Sediment, and 

Fish from English Lakes”, Environmental Science and Technology, 43, 9077–9083 (2009a).

(6) S. Harrad, M. A. Abdallah, A. Covaci “Causes of variability in concentrations and diastereomer patterns of Hexabromocyclododecanes in 

indoor dust”, Environment International, 35, 573–579 (2009b).

(7) F. Barontini, V. Cozzani, L. Petarca, “Thermal stability and decomposition products of hexabromocyclododecane”, Ind. Eng. Chem. Res., 40, 

3270–3280 (2001).

(8) Brandsma, S. H.; van der Ven, L. T. M.; de Boer, J.; Leonards, P. E. G. Identification of hydroxylated metabolites of 

hexabromocyclododecane in wildlife and 28-days exposed Wistar rats. Environ. Sci. Technol. 2009, 43 (15), 6058−6063. 

(9) Esslinger, S.; Becker, R.; Maul, R.; Nehls, I. Hexabromocyclododecane enantiomers: Microsomal degradation and patterns of hydroxylated 

metabolites. Environ. Sci. Technol. 2011, 45 (9), 3938−3944.
15

(10) Hakk, H.; Szabo, D. T.; Huwe, J.; Diliberto, J.; Birnbaum, L. S. Novel and distinct metabolites identified following a single oral dose of

alpha- or gamma-hexabromocyclododecane in mice. Environ. Sci. Technol. 2012, 46 (24), 13494−13503.

(11) Weber M, Durmaz V, Becker R, Esslinger S. Predictive Identification of Pentabromocyclododecene (PBCD) Isomers with High Binding 

Affinity to hTTR. Organohalogen Compounds, 71: 384-389 (2009).

(12) Gerecke, AC. Giger, W, Hartmann, PC, Heeb, NV, Koher, HP, Schmid, P, Zennegg, M, Kohler, M. Anaerobic Degradation of brominated 

flame retardants in sewage sludge, Chemosphere, 2006, 64, 311-317.

(13) Heeb, NV, Wyss, SA, Geueke, B, Fleischmann, T, Kohler HP, Lienemann, P. LinA2, a HCH-converting bacterial enzyme that 

dehydrohalogenates HBCDs. Chemosphere, 107, 194-202 (2014).

(14) Heeb NV, Zindel D, Graf H, Azara V, Schweizer WB, Geueke B, Kohler HP, Lienemann P. Stereochemistry of LinB-catalyzed 

biotransformation of -HBCD to 1R,2R,5S,6R,9R,10S-pentabromocyclododecanol. Chemosphere, 90, 1911-1919 (2013).

(15) Heeb NV, Zindel D, Geueke B, Kohler HP, Lienemann P. Biotransformation of Hexabromocyclododecanes (HBCD) with LinB – an HCHconverting bacterial enzyme. Environ. Sci. Technol., 46, 6566-6574 (2012).

(16) Heeb NV, Zindel D, Schweizer WB, Lienemann P. 2,5,6,9,10-Pentabromocyclodecanols (PBCDOHs): a new class of HBCD transformation 

products. Chemosphere, 88, 655-662 (2012).

(17) Heeb NV, Graf H, Bernd Schweizer W, Lienemann P. Isobutoxypentabromocyclododecanes (iBPBCDs): a new class of polybrominated 

compounds. Chemosphere, 78, 950-957 (2010).

EPA Response: EPA evaluated these references and added those that were determined to be 

relevant to the assessment. Only numbers 11, 16, and 17 were not added to the assessment. EPA 

also added several other references by N. V. Heeb.

Peer Reviewer 3 Comment: I have a number of recommendations with respect to the Chemistry 

and Environmental Fate section of the review:

The chemical structure of HBCD, particularly the structures of three predominant diastereomers, is 

not strongly emphasized, yet it is the significant differences in structures of the isomers through 

which differences in chemical properties are manifest, e.g., the roughly 40-fold difference in 

solubility between the alpha and gamma compounds. In fact, the summary includes only the generic 

2D structure of HBCD. The review does cite the 3D structures of Heeb et al. (2005), but I would 

also recommend referring to the work of Arsenault et al. 2007 (Chemosphere, Vol. 67, pgs. 1684-

1694) who structurally characterized the HBCD compounds in three dimensions by NMR 

spectroscopy. Undoubtedly, the structures of the individual HBCD diastereomers profoundly 

influence environmental fate. Overall, the review lacks isomer-specific discussion with respect to 

environmental processes.

EPA Response: EPA added more discussion about the isomers and included the suggested

reference. For example:

“Table 2-1 provides selected chemical and physical properties for HBCD. For many endpoints, such 

as water solubility and melting point, multiple value ranges are reported in the scientific literature. 

The differences in reported values are due to differences among technical mixtures of HBCD, which 

contain varying ratios of HBCD diastereomers. In addition to data reported for unspecified 

mixtures, the unique property values for α-, β-, and γ-HBCD diastereomers were reported, when 

available. For example, there is a >10-fold difference in water solubility between α- and γ-HBCD. It 

has been hypothesized that the measurable differences in properties are a result of differences in the 

three-dimensional physical structures of HBCD. The physical structures, shown in Figure 2-1, were 
16

calculated using a semi-empirical method with molecular modeling using the global minimum 

energy conformer, 1H nuclear magnetic resonance (NMR) and 13C NMR data (Arsenault et al., 

2007).”

Peer Reviewer 3 Comment: There is little discussion related to beta HBCD. Although this 

diastereomer can comprise over 10% of some technical mixtures, the reason(s) for its rare/trace 

occurrences in environmental compartments remain unclear. This fact should be mentioned in the 

review, as this observation is relevant to the potential interconversion of the HBCD isomers.

EPA Response: EPA added the suggested observation as follows:

“Although β HBCD can constitute over 10% of some technical mixtures, it is inconsistently found 

in environmental compartments in monitoring studies; the reason for this is not apparent in the 

available literature (NICNAS, 2012).”

Peer Reviewer 3 Comment: The delta and epsilon meso forms of HBCD are mentioned on page 

9. Although they only contribute sub-percent levels to some technical mixtures, it should be noted 

that these compounds are rarely, if at all, detected in environmental compartments.

EPA Response: EPA revised the text to reflect that these forms are rarely detected in 

environmental compartments.

“Small amounts of δ-HBCD (0.5%) and ε-HBCD (0.3%) have also been reported and may be due to 

impurities in the starting material (Heeb et al., 2005). δ- and ε-HBCD are tentatively regarded as 

meso forms, as chirality has not been detected for these stereoisomers. Although δ and ε-HBCD 

are rarely detected in environmental compartments, one monitoring study reported detection of δHBCD in 13 fish samples from English lakes, but not the associated water or sediment samples 

(Harrad et al., 2009b). Consistent with this finding, δ-HBCD has been suggested to be a 

biotransformation product in fish. Trout S9 fraction assays detected the production of δ-HBCD in 

samples containing only α-, β-, or γ-HBCD (Abdallah et al., 2014).”

Peer Reviewer 3 Comment: The review heavily cites the excellent review by Covaci et al. (2006) 

in Environmental Science and Technology. This is certainly an excellent source of information on 

the occurrence, distribution and fate of HBCD in the environment. A follow up to this review, with 

Dr. Covaci as a co-author, was published by Marvin et al. in Environmental Science and 

Technology in 2011 (Vol. 45, pgs. 8613-8623); this review focused on the increased understanding 

of HBCD in the environment based on the roughly 265 additional scientific papers published 

subsequent to the review by Covaci et al up to 2011. This ancillary review included discussions of 

physical – chemical properties, persistence, bioaccumulation, long-range transport potential, 

occurrence, distribution, temporal trends, human exposure and toxicity. The supplementary 

information (SI) for this review includes a comprehensive list and assessment of physical property 

data on HBCD.

EPA Response: Information from the review by Marvin et al. was included in the assessment in 

the appropriate sections.
17

Response to External Peer Review Section II: Human Health Effects

I. Charge to Reviewers

1. Does the summary statement and review of the literature present a clear rationale and 

accurately reflect the conclusions regarding the human health hazards of HBCD?

2. a) Please comment on the non-cancer endpoints. Has the scientific evidence for non-cancer 

endpoints been accurately and clearly represented and evaluated? Do you agree or 

disagree with the conclusions?

b) Please comment on the adequacy of the conclusions in the HBCD hazard assessment with 

regard to toxicity in the liver and thyroid.

c) Please comment on the adequacy of the conclusions in the HBCD hazard assessment with 

regard to neurotoxicity.

d) Please comment on the adequacy of the conclusions in the HBCD hazard assessment with 

regard to other developmental and reproductive effects.

3. Do you agree or disagree with the selection of the LOAELs/NOAELs (or BMDLs/BMDs) 

for all endpoints?

4. Are you aware of any additional key published studies or publicly available scientific reports 

that are missing from the draft document that might be useful for discussion of the hazards? 

If so, please provide a copy of the study or a complete citation.

5. Please provide any additional comments or suggestions you may have that may improve this 

section of the document.

II. Response to Human Health Hazard Assessment Charge

The reviewers of the human health hazard provided a portion of comments that were editorial in 

nature. These comments were considered by the U.S. EPA and addressed in the document where 

appropriate, but were not included in this response section.

A. General Impressions

None were provided by the peer reviewers

B. Response to Charge Questions

1. Does the summary statement and review of the literature present a clear rationale and 

accurately reflect the conclusions regarding the human health hazards of HBCD?

Peer Reviewer 4 Comment: The reviewer agrees to the conclusion that “there is sufficient 

evidence to support a concern for moderately high to high toxicity of HBCD.” Nonetheless, the 
18

major effect of HBCD, developmental neurotoxicity, is missing in the conclusion. The overall 

conclusion should also include the findings on acute toxicity, carcinogenicity, genotoxicity, the 

mode of action, skin sensitization, eye irritation, etc.

EPA Response: These findings are mentioned in the executive summary. The relevant text is cited 

below:

“Neurodevelopmental effects on behavior and learning were also observed in the literature

(Saegusa et al., 2009; Eriksson et al., 2009).”

“Studies evaluating the carcinogenic potential of HBCD in humans or animals were not available so 

no determination of carcinogenicity could be made. HBCD, however, does not appear to be 

genotoxic.”

“HBCD was not found to be highly toxic in acute oral, inhalation, and dermal tests in rodents.”

Peer Reviewer 4 Comment: Please describe the test substance used in the studies, in terms of the 

source and the composition of major isomers, when available. This is important for two reasons: (a) 

isomers have different distribution, accumulation, and rates of clearance in the body, and (b) the 

composition in the industrial grade HBCD is significantly different from the compositions of HBCD 

in food and other environmental media. Most of the existing toxicity data are derived from the 

industrial grade HBCD. The composition of the isomers can impact on toxicity. 

EPA Response: The isomeric composition of the test compound was included for all studies for 

which this information was available. This information was not available for all studies in this 

assessment. Some examples of these descriptions are below:

“In a subacute study, Van der Ven et al. (2006) exposed Wistar rats (5/sex/dose) by gavage to a 

mixture of HBCD dissolved in corn oil at concentrations resulting in doses of 0.3, 1.0, 3.0, 10, 30, 

100, and 200 mg/kg-day for 28 days. The isomeric composition of the HBCD was 10.3% α, 8.7% β, 

and 81.0% γ.”

“In a one-generation study that included additional immunological, endocrine and 

neurodevelopmental endpoints, van der Ven (2009) exposed Wistar rats (10/sex/dose) to a 

composite mixture of technical-grade HBCD in the diet at concentrations resulting in doses of 0.1, 

0.3, 1.0, 3.0, 10, 30, or 100 mg/kg-day. This study followed OECD Guidelines 415 and 407 

(OECD, 1983; OECD, 1995). The isomeric composition of the HBCD was 10.3% α, 8.7% β, and 

81.0% γ.”

“In a two-generation reproductive toxicity study, Ema et al. (2008) administered HBCD to 

Crl:CD(SD) rats conducted according to OECD Guideline 416 (OECD, 2001), U.S. EPA 

Guidelines (U.S. EPA, 1991; 1996), and Good Laboratory Practices. Groups of male and female 

rats (24/sex/dose) were fed HBCD (as a mixture of α-HBCD, β -HBCD, and γ-HBCD with 

proportions of 8.5, 7.9, and 83.7%, respectively) in the diet at concentrations of 0, 150, 1,500, or 

15,000 ppm from 10 weeks prior to mating through mating, gestation, and lactation.”

Peer Reviewer 4 Comment: Page 2: “HBCD was not found to be toxic in acute oral, inhalation, 
19

and dermal tests in rodents.”: This claim is not true. Please delete or modify this sentence. 

EPA Response: This statement was modified to “HBCD was not found to be highly toxic in acute 

oral, inhalation, and dermal tests in rodents.”

Peer Reviewer 4 Comment: Page 2: “In short-term and subchronic studies, HBCD demonstrated 

effects mainly on the thyroid and liver, predominately in females”: Thyroid hormone disruption 

should not be described as a “mainly” organ effects, other organ/system/tissue effects related to 

thyroid function are observed. In addition, many effects of HBCD have different sensitivity and 

manifestation in males and females. The phrase “predominately in females” is not true.

EPA Response: The phrases “mainly organ effects” and “predominately in females” were deleted 

from the report.

Peer Reviewer 4 Comment: P. 20, Line 10: Because of the significant sex differences, reporting 

the LD50 of 1258 mg/kg for male CD rats and LD50 of 680 for female CD rats is more meaningful 

than reporting the average 924 mg/kg. 

EPA Response: The LD50 was broken out by sex in the report. 

Peer Reviewer 4 Comment: P. 20, Paragraph 3, Line 17-21: Please consider an additional 

statement, in contrast to the Line 19-21 statement. Reviewer’s suggestion: “On the other hand, 

developmental behavioral defects have been observed in 3-month old mice after a single oral 

exposure on PND 10 (Eriksson et al., 2006).”

EPA Response: The suggested text was added to the report.

Peer Reviewer 4 Comment: P. 20, Line 20: please state the length of the recovery period. 

EPA Response: The recovery period was added to the report.

Peer Reviewer 4 Comment: P. 21, Line 3: Please consider making the change: “A” few 

reproductive effects...., 

EPA Response: The suggested editorial change was made in the report. 

Peer Reviewer 4 Comment: P. 22: Please make correction. Rats in the study by Lilienthal et al. 

(2009) were exposed premating, during mating, during gestation, and after birth, not “in utero” 

only, as it is stated on this age. 

EPA Response: The text was changed to the following: “In a study of HBCD effects on the 

dopaminergic system and auditory functions, Lilienthal et al. (2009) reported neurobehavioral 

effects of HBCD based on decreased latencies in haloperidol-induced catalepsy tests and elevated 

thresholds in frequency-specific stimuli tests in Wistar rats exposed during premating, mating, 

gestation, and after birth.”

Peer Reviewer 4 Comment: P. 22, Lilienthal (2009) cited on the page is missing in the list of 
20

References.

EPA Response: The citation was added to the list of references.

Peer Reviewer 4 Comment: Please consider removing/modifying the sentences in the Executive 

Summary, “...identity of HBCD metabolites and pathway for HBCD metabolism have not been 

established”, and similar statements on P. 20 and P. 23. (Note, Existing data do provide important 

information on the HBCD metabolic pathways.)

EPA Response: This statement was removed from the report. 

Peer Reviewer 4 Comment: P. 21-22, Ema et al. (2008): In the light of neurobehavioral effects of 

maternal hypothyroidism on the offspring in humans and laboratory animals, and the endocrine 

organs’ compensatory responses to maintain the thyroid function in the offspring, the significant 

increases in testis, brain and pituitary gland weights in F1 adults (Table 6 and Table 7 in Ema et al., 

2008) should not be dismissed. The 150 ppm treatment, therefore, is LOAEL. 

EPA Response: While there was a significant increase in relative testis weight in F1 weanlings at 

150 ppm, the percent change was relatively small (approx. 8.7%) and neither absolute nor relative 

testis weights were significantly changed at any dose group in F1 adults. In fact, relative testis 

weights in F1 adults were slightly, but not significantly, decreased compared with the control group.

While relative brain weight and relative pituitary gland weight were significantly increased in F1 

adults at 150 ppm, relative weights were not significantly increased in the higher dose groups (1,500 

ppm and 15,000 ppm). Due to the lack of a clear dose-response relationship, EPA did not conclude 

that 150 ppm should be the LOAEL based on these endpoints. 

Peer Reviewer 4 Comment: P. 23-29, Section 3.2.: Please consider the inclusion of the 

observations from additional articles on the differences in toxicokinetics of the three major isomers. 

Here are three additional publications: Hakk et al. (2012), Szabo et al. (2011a), and Szabo et al. 

(2011b).

EPA Response: These papers were added to the report. The added text is below:

“In a more recent study, α-HBCD and γ-HBCD produced distinct metabolites after a single 

administration of HBCD to C57BL/6 female mice (Hakk et al., 2012).”

“In a follow-up study, approximately 90% of α-HBCD administrated was absorbed after exposure 

(Szabo et al., 2011b).”

“In a similar study, an administered dose of α-HBCD was found primarily in adipose, liver muscle, 

and skin (Szabo et al., 2011b). Accumulation was dose dependent with nonlinear accumulation at 

higher doses. In a study investigating the differences in tissue distribution of α- and γ-HBCD 

between adult and developing mice, the authors reported that while distribution patterns were 

similar between adult and developing mice, concentrations of each diastereomer were higher in the 

pup’s liver, fat, kidney, brain, blood, muscle, and lungs than the adult’s (Szabo et al., 2011a). This 

finding suggests that developmental stage exposure to α- and γ-HBCD may result in increased body 

burden compared with adults.”
21

Peer Reviewer 4 Comment: See Brandsma et al. (2009) and Hakk et al. (2012) for the hydroxyl 

metabolites. 

EPA Response: These papers were added to the report. The added text is below:

“In another study, four different groups of hydroxylated HBCD metabolites were identified in rat 

adipose, liver, lung, and muscle tissue by liquid and gas chromotography combined with mass 

spectrometry: monohydroxy metabolites of HBCD, pentabromocyclododecene (PBCDe), 

tetrabromocyclododecene (TBCDe), and dihydroxy-HBCD (Brandsma et al., 2009).”

“In a more recent study, α-HBCD and γ-HBCD produced distinct metabolites after a single 

administration of HBCD to C57BL/6 female mice (Hakk et al., 2012).”

Peer Reviewer 4 Comment: Hakk et al. (2012), Szabo et al. (2010), and Szabo et al. (2011b) are 

useful in describing the stereoisomeriztion of HBCD. The article by Hakk et al. (2012) shows a 

proposed metabolic schemes in mice. Steroisomeric shift results in a proportionally higher human 

exposure to alpha-HBCD. Higher amounts of alpha-HBCD accumulation in human body has been 

observed, when the relative amounts of accumulation of isomers were compared with the 

composition of the technical-grade HBCD. Furthermore, the fact that alpha-HBCD can reach a 

higher concentration in the brain than an equal dose of gamma-HBCD, makes stereoisomerization 

an important metabolic pathway.

See articles by Sanders et al. (Sanders et al., 2013) and by Szabo et al. (2010, 2011), for the 

elimination half-lives of the three major isomers, metabolism of the isomers, and the pattern of 

isomer accumulation in tissues of mammalian species.

EPA Response: Szabo et al., (2010) is already cited in the report, but the Sanders et al., (2013) 

paper was added to the report. Below is the text added to the report:

“The extent of metabolism of β-HBCD in C57BL/6 female mice was found to be similar to that of 

γ-HBCD (Sanders et al., 2013).”

“The authors observed no dose dependency in fecal or urinary elimination of γ-HBCD. The extent 

of excretion of β-HBCD was found to be similar to that of γ-HBCD (Sanders et al., 2013).”

The text added to the report from Szabo et al. (2011a; 2011b) and Hakk et al. (2012) is included in 

previous responses above. 

Peer Reviewer 5 Comment: Based on the available data, the EPA has concluded that there is 

sufficient evidence to support a concern for moderately high to high toxicity of HBCD. The 

Technical Assessment Report of HBCD is well written and comprehensive, and provides a clear 

rationale for concern over the toxicity to humans, particularly with developmental exposure.

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 6 Comment: This summary provides an overview of the human health hazard 
22

assessment but it does have a large emphasis on the results from reproductive/developmental which 

could be condensed to be in keeping with the other results. Under 3.2.2 Distribution, there are many 

more recent papers on HBCD in human tissues. It is unclear from this section, if these papers were 

reported as examples of HBCD in human tissues or if perhaps the review was done in 2008/2009, I 

noted that some of the online references were accessed in 2008 or 2009 (but then others were 

accessed in January 2014). Is it possible to clarify the parameters of the review, that is, review all 

available data from xxx date to xxxx date? 

EPA Response: Given the time between the development of the assessment and the initiation of 

the peer review some newer studies may not have been covered in the draft assessment. However, 

EPA has added several additional studies to the assessment in response to the peer review 

comments. Specific papers are addressed in charge question 4. 

Peer Reviewer 6 Comment: Still in Section 3.2.2, is it possible for the units of measure to be the 

same. I understand the difficulty with this as different authors use different measures. If not possible 

to convert all to ng/g lipid, perhaps add a note to alert the reader to the change in units. Otherwise, I 

find that the summary statement and review of the literature are accurate.

EPA Response: All units were converted to ng/g lipid (or fat) where possible. The revised text is 

below:

“Antignac et al. (2008) analyzed samples from 26 mother-newborn pairs in France for the presence 

of brominated flame retardants. α-HBCD was detected in approximately half of the adipose samples 

(1 – 3 ng/g lipid weight) and in 7 of the breast milk samples (2.5 – 5 ng/g lipid weight). No β- or 

γ-isomers were detected in any of the samples. The authors were unable to measure HBCD in serum 

samples, however, due to analytical limitations. Weiss et al. (2006) reported HBCD (mostly the αHBCD diastereomer but also γ-HBCD) concentration in human serum from 53 wives of Swedish 

fisherman (identified by the authors as a high exposure group) ranging from <0.24 – 3.4 ng/g fat 

with a median concentration of 0.46 ng/g fat (n=50). In a study of Japanese women, α-HBCD (0.43 

– 1.9 ng/g lipid weight) was detected in all 11 pooled breast milk samples collected between 1988 

and 2006, γ-HBCD (0.24 – 2.60 ng/g lipid) was detected in 7 of the 11 samples, and β-HBCD was 

not detected (<0.2 ng/g lipid) (Kakimoto et al., 2008). Abdallah and Harrad (2011) measured 

HBCD in 34 human milk samples from Birmingham, UK. HBCD was detected in all samples and 

the average concentration of all HBCD isomers was 5.96 ng/g lipid weight. α-HBCD comprised 62 

– 95% of HBCD detected in the study, while β-HBCD and γ-HBCD comprised 2 – 18% and 3 –

33% of HBCD detected in the study, respectively. In an analysis of HBCD concentrations in sera 

from the Canadian Health Measures Survey, researchers detected HBCD in all samples analyzed 

(Rawn et al., 2014). In most samples, HBCD was detected as <1 ng/g lipid, similar to global 

concentrations. α-HBCD was the biggest contributor to total HBCD, although β-HBCD and 

γ-HBCD were detected in 23% and 35% of the samples, respectively. No differences in total HBCD 

concentration were associated with age or sex.”

Peer Reviewer 6 Comment: In the first 2 paragraphs is it possible to include references for the 

statements made? I understand sometimes summaries do not contain references but further on 

references are used and so it makes sense to use them in the first two paragraphs also. It makes it 

difficult to determine the source data without the references.
23

EPA Response: References were added to the summary sections.

Peer Reviewer 6 Comment: First sentence of the summary “...including fatty tissues....” Could 

you add in here that HBCD is detected in human milk and human serum. There is no mention in the 

summary of detection in human samples.

EPA Response: This has been added to the summary. The revised summary text is below:

“HBCD is absorbed via the gastrointestinal tract and metabolized in rodents. Once absorbed, HBCD 

is distributed to a number of tissues, including fatty tissue, muscle, and the liver, where it 

accumulates unchanged. Elimination of HBCD is predominantly via feces (as the parent 

compound), but it is also eliminated in urine (as secondary metabolites). HBCD has been detected 

in human milk and serum.”

Peer Reviewer 6 Comment: From a reader’s perspective, I find the placement of the reference 

makes reading the text somewhat difficult. For example on page 25 the Reistad reference is in the 

middle of the text about this reference. Is the text below this reference still talking about Reistad or 

the next reference?

EPA Response: The text was modified to address this issue. The revised text is below:

“Studies on the distribution of HBCD in rodents have measured HBCD concentrations in organs 

such as the liver, kidneys, and brain. HBCD was identified in the liver (1,248 ± 505 μg/kg, w/w) 

and brain tissue (49 ± 13 μg/kg, w/w) of 3 male Wistar rats 72 hours after a single intraperitoneal 

(i.p.) injection of 18.7 ± 0.7 mg HBCD/kg (Reistad et al., 2006).”

Peer Reviewer 6 Comment: At 3.2.4 Excretion – should it be clarified how Geyer estimated halflife? He used 2 different methods based on literature and no human data.

EPA Response: The text was modified to address this issue. The revised text is below:

“In humans, Geyer et al. (2003) estimated the HBCD total body half-life and the terminal half-life 

from adipose tissue as 64 days (with a range of 23 – 219 days). The total body half-life was 

estimated using daily intake and total body burden under steady state conditions in nonoccupationally exposed adult humans according to a one compartment, open pharmacokinetic 

model. The terminal half-life was estimated from applying an interspecies adjustment to the 

terminal half-life in rats found in the literature. No other human data on elimination of HBCD were 

found in the literature.”

Peer Reviewer 6 Comment: At 3.2.4 Excretion – elsewhere the text lists excretion in urine and 

faeces separately but for Szabo et al., it is combined rather than 50% faeces and 30% urine.

EPA Response: The text was modified to address this comment. The revised text is below:

“Szabo et al. (2010) observed that approximately 80% of an administered dose of γ[14C]-HBCD was 

eliminated in the urine (30%) and feces (50%) four days following a single gavage dose of 3 

mg/kg.”
24

2a. Please comment on the non-cancer endpoints. Has the scientific evidence for non-cancer 

endpoints been accurately and clearly represented and evaluated? Do you agree or disagree 

with the conclusions?

Peer Reviewer 4 Comment: Improvements are needed. The reviewer provides the following 

suggestions for additional information, clarification, and corrections. 

There is a dermal study published in Japanese (Momma et al. (1993), on skin sensitization. 

EPA Response: EPA added the Momma et al., 1993 study to the assessment. Below is the added 

text:

“Dermatological Evaluation of a Flame Retardant, Hexabromocyclododecane (HBCD), on Guinea 

Pig by Using the Primary Irritation, Sensitization, Phototoxicity, and Photosensitization of Skin 

(Momma et al., 1993)

The skin sensitization test was carried out according to the maximization test method of 

Magnusson and Kligman. HBCD was dissolved in olive oil to a concentration of 0.05, 0.5, or 5%. 

When induction of sensitization occurred, challenge doses of 0.005, 0.05, 0.5, or 5% (dissolved in 

acetone) were applied. The induction dose of greater than 0.5% and the challenge dose greater than 

0.05% elicited a positive response. The increase in the concentration of induction and challenge 

doses did not further increase the percentage of positive responses or the intensity of response. The 

authors concluded that HBCD is a mild skin allergen.”

Peer Reviewer 4 Comment: P.24, Line 4-7: Please indicate the total sample size (Weiss et al., 

2006), and the number of samples used for the range “0.3-5.4 pmol/g”, and median. 

EPA Response: These modifications were made to the report. The revised text is below:

“Weiss et al. (2006) reported HBCD (mostly the α-HBCD diastereomer but also γ-HBCD) 

concentration in human serum from 53 wives of Swedish fisherman (identified by the authors as a 

high exposure group) ranging from <0.24 – 3.4 ng/g fat with a median concentration of 0.46 ng/g 

fat (n=50).”

Peer Reviewer 4 Comment: P.24, Line 15-16: Please enter n=69 for maternal serum and n=12 for 

cord serum.

EPA Response: These sample sizes were added to the report. The revised text is below:

“Researchers have also found that HBCD can be transferred through the placenta during critical 

stages of infant development (Meijer et al., 2008). These authors detected HBCD in the maternal 

serum (n=69) and cord serum (=12) of pregnant women and their infants in the 35th week of 

pregnancy.”

Peer Reviewer 4 Comment: P. 26, Paragraph 2, line: (a) The publication by van der Ven et al. 

(2006), cited in this sentence is missing on page 66. 
25

EPA Response: The reference was added to the reference list.

Peer Reviewer 4 Comment: P. 26, For the sentence., “The isomeric composition of 

HBCD....technical compound”: It is important to note that in the study by van der Ven et al. (2006), 

the animals were exposed orally for 28 days, beginning at the age of 7 wks, while the animals in the

study of van der Ven et al. (2009) were exposed through maternal gestational and locational 

exospore. The concentration and composition of the three isomers in the two studies should be 

presented in the context of two different treatment methods.

EPA Response: The text was modified to address this comment. The revised text is below:

“The isomeric composition of HBCD detected in the livers of Wistar rats following a 28-day gavage 

study with exposure beginning at the age of 7 weeks (van der Ven et al., 2006) and a dietary 

reproduction study with exposure occurring during gestation and lactation (van der Ven et al., 2009) 

was different from the isomeric composition of the administered technical compound. Much lower 

ratios of γ-HBCD to α-HBCD were observed in the liver compared with the administered 

compound.”

Peer Reviewer 4 Comment: P. 26, Paragraph 2: Some of the information in this paragraph can be 

used to illustrate the metabolism of HBCD in Section 3.2.3.

EPA Response: The following text was moved to the metabolism section:

“The isomeric composition of HBCD detected in the livers of Wistar rats following a 28-day gavage 

study with exposure beginning at the age of 7 weeks (van der Ven et al., 2006) and a dietary 

reproduction study with exposure occurring during gestation and lactation (van der Ven et al., 2009) 

was different from the isomeric composition of the administered technical compound. Much lower 

ratios of γ-HBCD to α-HBCD were observed in the liver compared with the administered 

compound. In van der Ven et al. (2009), the concentration of α- and γ-HBCD in the liver (expressed 

as μg HBCD/g lipid) of F1 males and females increased in a dose-related fashion. β-HBCD was 

only detected at the highest exposure concentrations. The ratio of γ/α isomers was 8.1 in the 

administered technical mixture of HBCD, but the ratio in the livers of exposed F1 animals ranged 

from 0.04 to 2.2 in females and from 0.2 to 1.4 in males. Concentrations were higher in females 

over the entire range of doses (four and six times higher for γ- and α- isomers, respectively, at the 

highest doses). In van der Ven et al. (2006), the average ratio between γ- and α-HBCD was 8.1 in 

the administered technical mixture, whereas the average ratio in the liver was 2.9 in females and 1.9 

in males. Females had higher HBCD concentrations in the liver, and the ratio of lipid-normalized γHBCD to α-HBCD in the liver decreased with increasing exposure concentration (liver lipid 

concentrations did not differ among dose groups) and the duration of oral exposure.”

Peer Reviewer 4 Comment: P. 27, Paragraph on CYP: This part is presented as effects of HBCD 

on cytochrome P450 enzymes. How is this related to the metabolism of HBCD? 

EPA Response: EPA agrees that this information is not related to the metabolism of HBCD. This 

paragraph was deleted from the report.

Peer Reviewer 4 Comment: Please consider the information in the article for calculated half-lives 
26

of HBCD isomers in humans: (Abdallah and Harrad 2011). 

EPA Response: This paper was added to the report. The added text is below:

“Abdallah and Harrad (2011) detected two HBCD degradation products for the first time in human 

tissues. The degradation products pentabromocyclododecenes (average concentration of 0.04 ng/g 

lipid weight; n=9) and tetrabromocyclododecadienes (average concentration of 0.15 ng/g lipid 

weight; n=25) were both detected in human milk samples from Birmingham, UK.”

Peer Reviewer 4 Comment: For tissue half-lives of gamma-HBCD in mice see (Szabo et al., 

2010). 

EPA Response: The following text was added to address the distribution of gamma-HBCD:

“Similar results were obtained in single oral dose toxicity studies. Following a single gavage 

exposure of γ-HBCD to female C57BL/6 mice, the largest percentage of the dose was observed in 

the liver, followed by the skin and muscle (Szabo et al., 2010).”

Peer Reviewer 4 Comment: For information on alpha- and gamma-HBCD see (Hakk et al., 2012).

EPA Response: The following text was added to the report:

“In a more recent study, α-HBCD and γ-HBCD produced distinct metabolites after a single 

administration of HBCD to C57BL/6 female mice (Hakk et al., 2012). In mice exposed to α-HBCD, 

four hydroxylated metabolites were detected in fecal extracts, and one of these metabolite isomers 

was found in liver, brain, and adipose tissue. Fecal extracts from mice exposed to γ-HBCD, 

however, contained multiple isomers of monohydroxy-pentabromocyclododecene, dihydroxypentabromocylcododecene, and dihydroxy-pentabromocyclododecadiene, while in liver and adipose 

tissue only a single monohydroxy-ptentabromocyclododecane metabolite was detected.”

Peer Reviewer 4 Comment: P. 35: The statements in Paragraph 1 over simplify the observed 

effects. Please provide adequate description of observed effects. It is important to include the effects 

that may be associated with recognized potential modes of actions of HBCD, which include the 

disturbance of thyroid hormonal balance and of the HP-thyroid axis. The last statement appears to 

emphasize the “effects not significant after a recovery period”, while the purpose of this section is 

to provide sufficient description of the observed effects. Please also see above Comment (3). 

EPA Response: While there is some evidence of this potential mode of action for HBCD, EPA 

cannot speculate on its potential link with the observed effects in this hazard assessment. The 

statement regarding the apparent reversibility of observed effects after the recovery period was

included to provide the reader with a complete account of the findings from the reviewed studies. 

Peer Reviewer 4 Comment: P. 34: Please clarify that rats were exposed daily by gavage for 28 

days, for study by van der Ven et al. (2006).

EPA Response: The text was modified to address this comment. The revised text is below:

“In a subacute study, Van der Ven et al. (2006) exposed Wistar rats (5/sex/dose) by gavage to a 
27

mixture of HBCD dissolved in corn oil at concentrations resulting in doses of 0.3, 1.0, 3.0, 10, 30, 

100, and 200 mg/kg-day for 28 days.”

Peer Reviewer 5 Comment: Detailed comments are given below. The scientific evidence for noncancer endpoints has been accurately represented and evaluated. I agree with the conclusions with 

the exception of a few minor points presented in the following sections.

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 6 Comment: From reviewing the references, the non-cancer endpoints have been 

accurately and clearly represented and evaluated and having reviewed the source documents I agree 

with the conclusions. 

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 6 Comment: In Table 3-2 for the IRDC studies “exposure” is listed as Firemaster, 

is it possible to list GLS-S6-41A in the exposure column for the Lewis/Palanker and Gulf South 

studies?

At Acute Oral Toxicity Studies (IRCD 1977), should it be “some males”, rather than “Males” as not 

all experienced these endpoints?

In Table 3-2, for Lewis and Palanker under Notes – it says “All rats gained weight over the 14-day 

observation period” whereas for IRDC it says “No...effects on body weight gain” but the document 

uses very similar wording as Lewis and Palanker “All rats continued to gain weight after exposure” 

and it would be easier to compare if this wording was used for IRDC section of the table also.

EPA Response: The table was modified to address these comments. The revised table is below:
28

2b. Please comment on the adequacy of the conclusions in the HBCD hazard assessment with 

regard to toxicity in the liver and thyroid.

Peer Reviewer 4 Comment: The effects on liver and thyroid were not adequately stated. 

P. 47: The effects on maternal liver and thyroid were not adequately stated. See original publication 

p. 461. 

P. 47, Serum T4 and TSH concentrations on PNW11 was not stated. 

P 47. Paragraph two: Information organization in this paragraph can be improved.

EPA Response: The text was modified to address these comments. The revised text is below:

“In parental rats, the relative weight of the thyroid and histopathological changes, including diffuse 

follicular cell hypertrophy, were significantly increased in females in the 10,000 ppm dose group. 

Changes were also observed in the other treatment groups, but these changes were not statistically 

significant. No changes in the number of implantation sites, number of live offspring, or male ratio 

were observed. 

Multiple changes were identified in the F1 generation. Selected findings are summarized in 

Table 3-3. No changes in body weight, anogenital distance, or onset of puberty in males or females 

were observed. At PND 20, statistically significant increases in relative liver weight and diffuse 

vacuolar degeneration of liver cells were observed in both males and females at 10,000 ppm. T3 

levels in males were significantly decreased and TSH levels significantly elevated at the highest 

dose, while T4 levels were unaltered at any dose. At PNW 11, the authors observed a significant 

dose-dependent increase in relative thyroid weights in male rats at 1,000 and 10,000 ppm. Relative 

liver weight was elevated in the 100 ppm dose group males, but changes were not observed at 

higher doses or in females at any dose level. Relative epididymides weight was also elevated in 100 

Table 3-2. Acute inhalation toxicity of HBCD and a residue of HBCD

Species/strain/test Exposure Result Notes Reference

Rat/Charles 

River/LD50

4-hr exposure to 

Firemaster 100 dust: 

202.14 mg/L

No mortality;

slight dyspnea at 

the end of 

exposure only

All rats gained weight over 

the observation period, but 

no control data were 

reported.

IRDC, 1977

Rat/Wistar/limit test 1-hr exposure to 200 

mg/L GLS-S6-41A

(highest possible 

concentration)

No mortality; no 

clinical signs

All rats gained weight over 

the observation period, but 

no control data were 

reported.

Lewis and Palanker, 

1978

Rat/Charles 

River/LC50 test of 

Firemaster 100 

residue (liquid)

4-hr exposure to 

Firemaster 100 

residue: 22.9 mg/L

One death (female, Day 3 post-exposure); 

dyspnea, salivation, lacrimation, and nasal 

discharge during exposure, resolved by Day 3; 

significant body weight loss through Day 3, but 

beginning to recover by Day 14 post exposure; 

signs of nasal and lung irritation at gross 

necropsy of rat that died.

IRDC, 1978b

Rat/NR/LC50 test 1 hr. whole-body 

inhalation exposure 

to GLS-S6-41A

LC50 >200 mg/L No significant effects 

observed.

Gulf South Research 

Institute, 1988
29

ppm dose group males, but changes were not observed at higher doses. Additionally, T3 levels 

were significantly decreased at 1,000 and 10,000 ppm compared to controls, but a dose-response 

relationship was unclear. Serum T4 levels and TSH concentrations were unchanged at PNW 11.”

Peer Reviewer 4 Comment: P. 47: “Onset of puberty in parental males and females was not 

affected.”: where does this come from?

EPA Response: The statement was deleted from the report.

Peer Reviewer 5 Comment: The study by van der Ven (2006) has been accurately summarized. 

This is a well-conducted study that identified a BMDL of 1.6 mg/kg/day (CED of 3.4 mg/kg/day) 

based on a 10% increase in thyroid weights in females. This study has been accurately summarized 

in the EPA report and I agree with the conclusions regarding the BMDL and the CED. 

Chengelis (1997, 2001) conducted a 28 day and a 90 day subchronic toxicity study in rats Both 

studies identified a NOAEL of 1000 mg/kg/day. Effects on clinical chemistry, hormone, organ 

weight and histology data were considered secondary to inducing effects on the liver or otherwise 

not considered adverse. The decrease in T4 levels and thyroid follicular hypertrophy should have 

been given more weight given the findings by van der Ven (2006). The latter investigator used more 

advanced technology and sophisticated data analysis, and the results should be given more weight 

than those of Chengelis (1997, 2001, 2002). I agree with the EPA conclusion that a LOAEL of 100 

mg/kg/day should be assigned based on liver weight and histopathological changes from the 90 day 

subchronic toxicity study. 

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 5 Comment: The Chengelis et al. (1997) document (EPA HPV Chemical 

Assessment) provided as a pdf includes a description of a 28 day and a 90 day subchronic toxicity 

study conducted in rats at BASF (Zeller and Kirsch, 1969, 1970). No mention was made of these 

studies in the EPA Technical Assessment document.

EPA Response: These papers were added to the assessment. The added text is below:

“In an older subacute study (Zeller and Kirsch 1969 as cited by U.S. EPA, 2001) an HBCD product 

no longer produced was administered to Sprague-Dawley rat (10/sex/group) at doses of 0, 1, 2.5, 

and 5% of the diet for 28 days. Doses were calculated to be 0, 940, 2410, 4820 mg/kg-day. No more 

details on study methodology were available. No clinical signs were observed at the 1% dose level 

and no significant change in mean body weight was observed at the 1 and 2.5% dose levels. Mean 

liver weight (both absolute and relative) was increased in all dose groups, but no microscopic 

pathology was detected. Thyroid hyperplasia was observed in some animals at all doses in addition 

to slight numerical development of the follicles and ripening follicles in the ovaries at the high dose. 

No gross or microscopic changes were detected in any other organ nor were clinical chemistry 

changes detected. The authors attributed the increased liver weight to hyperactivity as a result of 

increased thyroid activity and concluded that the increased liver weights were not pathologic. They 

identified a NOAEL or 940 mg/kg-day.”
30

“In an older subchronic study (Zeller and Kirsch 1970 as cited by U.S. EPA, 2001) an HBCD 

product no longer produced was administered to Sprague-Dawley rat (10/sex/group) at doses of 0, 

0.16, 0.32, 0.64, and 1.28% of the diet for 90 days. Doses were calculated to be 0, 120, 240, 470, 

and 950 mg/kg-day. Doses up to 0.64% produced no adverse clinical signs, no change in body 

weight, and no change in clinical chemistry results. An increase in relative liver weight was 

observed and was accompanied by fatty accumulation. The pathology report concluded that 

although fat was visible microscopically in treated rats, the change was not accompanied by any 

pathology, and therefore could not be defined as “fatty liver”. No histological changes were found 

in any other organ. The authors concluded that the increased liver weight and the fat deposits, both 

of which were largely reversible when administration of HBCD was stopped, were the result of a 

temporary increase in the activity of the liver. They identified a NOAEL of 950 mg/kg-day.”

Peer Reviewer 6 Comment: Again, from a reader’s perspective, it is somewhat difficult to find 

the Liver/Thyroid toxicity, in comparison to the Developmental and Reproductive toxicity which 

has a separate heading. After reviewing the source data, I agree with the conclusions with regards to 

toxicity in the liver and thyroid. 

EPA Response: As the liver and thyroid effects were considered developmental effects, they were 

included under the Developmental and Reproductive Toxicity heading.

Peer Reviewer 6 Comment: Some comments: I may have missed something but the Van den Van 

paper doesn’t seem to mention a dose of 200 mg/kg-day but this is listed on page 35 of the review. 

No, I now realize. The reference in the text says Van den Ven et al. (2006) but the one in the 

reference list is (2009). The 2006 paper does mention 200 mg/kg-bw. This error in the reference list 

should be corrected. 

EPA Response: The reference list has been corrected. 

Peer Reviewer 6 Comment: It would be easier to read if all the female observations were kept 

together and all the males ones kept together

EPA Response: EPA determined that grouping the findings by endpoint, rather than by sex

provides a clearer presentation of the data. 

Peer Reviewer 6 Comment:

There is a typographical error at the top of page 37. “non-caner” should be “non-cancer”

On page 37, the citation is Chengelis 1997 as cited by US EPA but when reading the US EPA 

document they cite Chengelis 1996.

EPA Response: The text has been modified to correct these typographical errors. 

Peer Reviewer 6 Comment: Is it possible to have a figure like 3-1 for other endpoints?

EPA Response: Figure 3-1 incorporates all of the endpoints from the chronic studies cited in the 

assessment. EPA does not believe that creating another figure for shorter term studies or mode-ofaction studies would add value to the assessment. 
31

2c. Please comment on the adequacy of the conclusions in the HBCD hazard assessment with 

regard to neurotoxicity.

Peer Reviewer 4 Comment: The conclusion with regard to neurotoxicity is insufficient. 

(1) The discussion on HBCD effects on dopamine-dependent behavior and hearing function is 

missing in Section 3.5. 

(2) A new subsection in Section 3.5, on developmental neurotoxicity, can be helpful. 

EPA Response: The Lilienthal et al. (2009) study regarding dopamine-dependent behavior and 

hearing function was not included in the Developmental and Reproductive Toxicity Section as it did 

not examine toxicity endpoints, but rather focused on exploring specific mechanisms of toxicity. 

EPA did not identify additional chronic studies on neurotoxicity to include in the assessment so a 

separate subsection was not created for the one developmental neurotoxicity study (Eriksson et al., 

2006).

Peer Reviewer 4 Comment: Additional comments:

(3) P. 55, Line 6: Please specify the dosing time, i.e., the “critical period of brain development” 

targeted in the study by Eriksson et al. (2006). 

(4) P. 60, Line 5: Please complete the sentence: “??? day –old F1 rats for haloperidol-induced 

catalepsy...”

EPA Response: The text was modified to improve clarity. The revised text is below:

“The potential effects of HBCD on behavior, learning, and memory were assessed in male mice that 

were exposed on PND 10 (Eriksson et al., 2006).”

“The general study design is described in the summary of van der Ven et al. (2009) in Section 3.5. 

Lilienthal et al. (2009) examined 110 day-old F1 rats for haloperidol-induced catalepsy to determine 

possible effects of HBCD on the dopaminergic system.”

Peer Reviewer 4 Comment: P. 60, Section 3.8 Potential Modes of Action: Please consider the 

following aspects in potential mode of action: (a) the effects on HBCD on metabolism enzymes, (b) 

fetal dependency on material thyroid hormone, and (c) the impact of thyroid hormone disturbance 

on offspring’s in-utero neuroendocrine development.

EPA Response: While there is some evidence of this potential mode-of-action for HBCD, EPA 

cannot speculate on its potential link with the observed effects in the context of this hazard 

assessment.

Peer Reviewer 5 Comment: Eriksson et al. (2006) administered HBCD at 0 (fat emulsion), 0.9 or 

13.5 mg/kg/day on PND10 and evaluated spontaneous behavior, learning and memory capacity in 

mice at 3 months of age. The authors identified a LOAEL of 0.9 mg/kg/day based on reduced 

locomotor activity compared to controls during the first 20 minutes of testing. The EPA, however, 

identified a LOAEL of 13.5 mg/kg/day based on decreased habituation locomotion and rearing in 

all intervals. I agree with the EPA assessment as the response at the high dose was more consistent 

across and within tests than at the lower dose.

See comments on the Lilienthal et al (2009) study in the next section.
32

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 6 Comment: After reviewing the source data, the summary is an accurate 

description, however, it seems there is only one study on neurotoxicity, is there more data? The text 

states that this study was not conducted according to current guidelines, I also notice no reference to 

ethical approval. Does this decrease the validity of the results? Under 3.8 Potential modes of action, 

a neurobehavioural study is reported. Can these two be put in the same section?

EPA Response: EPA did not identify additional chronic studies on neurotoxicity to include in the 

assessment. While a non-guideline study does not carry as much weight as a guideline study, EPA 

does not automatically dismiss the findings of non-guideline studies. The validity of a study is 

determined on a case-by-case basis, taking into account several factors. The Lilienthal et al. (2009) 

study was not included in the Developmental and Reproductive Toxicity Section with the one 

developmental neurotoxicity study as it did not examine toxicity endpoints, but rather focused on 

exploring specific mechanisms of toxicity. 

2d. Please comment on the adequacy of the conclusions in the HBCD hazard assessment with 

regard to other developmental and reproductive effects.

Peer Reviewer 4 Comment: There are conclusion statements at the end of discussions for each 

developmental/ reproductive study, but there is no overall conclusion or summary on developmental 

and reproductive effects.

EPA Response: A brief discussion of the developmental and reproductive studies is included in the 

executive summary (Section 1) and in the human health summary (Section 3.1). EPA believes that 

that these summaries are sufficient for the purposes of this hazard assessment.

Peer Reviewer 5 Comment: Murai et al. (1985) conducted a developmental toxicity study with 

HBCD given in the diet at concentrations of 0, 0.01, 0.1 or 1% throughout gestation (GD 0-20). The 

major findings were decreased food consumption and increased liver weight in the high dose group. 

The litter was not used as the experimental unit of analysis in this study. Otherwise, this appears to be 

a limited study which did not include measurements of the thyroid axis. I am not able to assess the 

re-calculations of the doses conducted by the European commission and thus the NOAELS and 

LOAELS due to lack of appropriate data.

EPA Response: The text was modified to improve clarity. The revised text is below:

“Female Wistar rats were fed HBCD in the diet at concentrations of 0, 0.01, 0.1, or 1% throughout gestation 

(Days 0 – 20) (Murai et al., 1985; Japanese with English abstract). Dams in the high-dose group had a 

statistically significant decrease (8.4%) in food consumption and increase in liver weight (13%) in 

comparison with controls. There were no treatment-related effects on maternal or fetal body weight. There 

were no effects on the number of implants; number of resorbed, dead, or live fetuses; body weight of live 

fetuses; or incidence of external or visceral abnormalities. A few skeletal variations were present but were 

also observed in controls and not considered significant. There were no effects on weaning or survival.

The European Commission (European Commission, 2008) used the study’s data to calculate the 

doses to be 0, 7.5, 75, and 750 mg/kg-day (based on the assumption of a mean animal weight of 

200g and food consumption of 15 g/day). They concluded that the offspring NOAEL was 750 
33

mg/kg-day and the maternal LOAEL was 750 mg/kg-day based on 13% liver weight increase in the 

high dose group."

Peer Reviewer 5 Comment: Ema et al. (2008) conducted a two-generation reproductive toxicity 

study on HBCD given in the diet at 0, 150, 1500 or 15,000 ppm. They identified a NOAEL of 150 

ppm (10.2 mg/kg/day) based on a decrease in the number of primordial follicles in the ovaries of F1 

females and decreased thyroid follicle size. The EPA has taken issue with this study for several 

reasons. One was that the systemic dose was uncertain as the animals were dosed with particles 

mixed in feed and particle size affects absorption rate. The investigators made no efforts to insure 

uniform particle size, but did state that HBCD was homogeneous in the diet and stable throughout 

the study. It is unclear what the basis of the EPA concern is, and more detail should be given on this 

point. Another concern on the part of the EPA was that the study did not consider litter effects, i.e., 

that the litter was not used as the unit of analysis. However, the authors state that data on offspring 

before weaning were statistically analyzed using the litter as the experimental unit, which is 

appropriate. Finally, the EPA identified a LOAEL of 150 ppm (~ 14 mg/kg/day) based on increased 

TSH levels in F0 females, a reproductive LOAEL of 1,500 pp, (~ 138 mg/kg/day) based on 

decreases in primordial follicle count and a developmental LOAEL 15,000 pm (~ 1142 mg/kg/day) 

based on increased pup mortality in the F2 generation. Ema et al. (2008) considered that the 

elevated TSH levels in F0 females at 150 ppm were not of toxicologic significance because they 

were not accompanied by histopathological changes in the thyroid gland nor changes in T4 levels 

and were not consistent across generations. I agree with the EPA position; TSH levels are the most 

sensitive indicator of thyroid hormone status, levels were clearly trending upward at 150 ppm in F2 

females, and they need not be consistent across generations to be important. 

EPA Response: EPA agrees with the comment, and the discussion of the litter not being used as 

the statistical unit in the Ema et al. (2008) was removed from the hazard assessment. The discussion 

of systemic doses in Ema et al (2008) was also removed from the hazard assessment. 

Peer Reviewer 5 Comment: Saegusa et al. (2009) evaluated the developmental toxicity of HBCD 

administered in the diet at doses of 0, 100, 1000 or 10,000 ppm from GD 10 to PND 20; offspring 

were evaluated up to PNW 11. They identified a maternal LOAEL of 10,000 ppm (~1505 

mg/kg/day) based on increased incidence of thyroid follicular cell hypertrophy, a developmental 

LOAEL of 1,000 ppm (~146 mg/kg/day) based on increased relative thyroid weight and decreased 

T3 levels in F1 males; a reproductive LOAEL was not identified. An important finding was reduced 

density of CNPase positive oligodendrocytes at 10,000 ppm, suggesting impaired oligodendroglial 

development due to developmental hypothyroidism. Deficiencies were that relatively small numbers 

of animals were studied, and thyroid hormone measurements were not made on female F1 rats, 

which have been found to be more sensitive to HBCD effects than male rats. The authors did 

consider litter effects as data on offspring during the lactation period were analyzed with the litter as 

the experimental unit of analysis. The LOAELS have been appropriately identified in the 

publication and in the EPA report.

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 5 Comment: van der Ven et al. (2009) conducted a one-generation study at dose 

levels of 0.1, 0.3, 1.0, 3.0, 10, 30 or 100 mg/kg/day. The most sensitive endpoints for female rats 

were decreased bone mineral density with a BMDL of 0.056 mg/kg/day and a decreased 
34

concentration of apolar retinoids in the liver with a BMDL of 1.3 mg/kg/day. The most sensitive 

endpoint for male rats was an increased IgG response to sheep red blood cells with a BMDL of 0.46 

mg/kg/day. There were no significant effects of HBCD exposure on any measure of reproduction, 

nor on any of a series of thyroid hormone measurements. The authors recalculated BMDLs as 

observed responses against internal liver concentrations per animals for effects detected at final 

necropsy; these recalculated BMLDs were 1.3 mg/kg/day for decreased bone mineral density in 

females and 44 mg/kg/day for decreased liver concentrations of apolar retinoids in females. In my 

opinion the original BMDLs and not the recalculated BMLDs should be used as normalization to 

liver concentrations is an untried and untested approach. This is an excellent study and the 

conclusions warrant concern for human health at relatively low exposure levels.

EPA Response: EPA agrees that the original BMDLs should be used, the following text was 

deleted from the report to improve clarity:

“These recalculated BMDLs were 1.3 mg/kg-day for decreased bone mineral density in females and 

44 mg/kg-day for decreased liver concentrations of apolar retinoids in females.”

Peer Reviewer 5 Comment: Lilienthal et al. (2009) reported the results of a neurobehavioral 

assessment from the one generation study conducted by van der Ven et al. (2009) described above. 

Haloperidol-induced catalepsy and brainstem auditory evoked potential (BAEP) tests were 

conducted on F1 offspring after 100 days of age. The authors concluded that results from the 

catalepsy test indicated reduced latencies to movement onset in female offspring, indicating effects 

on dopamine-dependent behaviors. The overall pattern of BAEP alterations suggested a 

predominant cochlear effect, possibly influenced by reduced levels of retinoids. Effects were dosedependent with BMDL values between < 1 and 10 mg/kg/day. Tissue concentrations at the BMDL 

values (reported in the van der Ven, 2009 study) were 3-4 orders of magnitude higher than current 

exposure levels in humans. Given that levels of HBCD are accumulating in human and aquatic 

populations, these findings are of great concern. However, given the experimental nature of the tests 

conducted, replication and further validation of the results are needed. In the EPA report, concerns 

were expressed that it was difficult to determine if the observed effects were due to developmental 

exposure to HBCD, a result of repeated-dose exposure or a combination of the two. These do not 

seem to be compelling concerns as both types of exposures occur in human populations.

EPA Response: The following text was removed from the report:

“It is difficult to determine if the observed effects were due to developmental exposure to HBCD, a 

result of repeated-dose exposure, or a combination of the two.”

Peer Reviewer 6 Comment: After reviewing the source data, the conclusions are an accurate 

summary of the source data with regard to other developmental and reproductive effects.

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 6 Comment: Some comments: On page 47, there is a paragraph on LOAEL, it is 

not clear from reading this text whether this was determined by the author of the text or was 

obtained from the Saegusa 2009 paper. This is not mentioned in the paper though. Rather, the 

NOAEL is discussed, but not mentioned in the text of this review, is there a reason? “Thus, based 
35

on the developmental brain effect, 100ppm was determined to be the no-observed-adverse- effect 

level (NOAEL) of HBCD from changes in thyroid parameters, translating into 8.1–21.3 mg/kg-d by 

maternal exposure level.”

EPA Response: The text was modified to improve clarity. The revised text is below:

“EPA identified a maternal LOAEL of 10,000 ppm (approximately 1,504.8 mg/kg-day) based on 

increased incidence of thyroid follicular cell hypertrophy. EPA identified a developmental LOAEL 

of 1,000 ppm (approximately 146.3 mg/kg-day) based on increased relative thyroid weight and 

decreased T3 levels in F1 male rats at PNW 11. Changes in reproductive endpoints (e.g., the 

number of implantation sites, live offspring, sex ratio) were not observed.”

Peer Reviewer 6 Comment: In the Ema et al. section, second paragraph, for clarity it should read 

“twenty-four male and 24 female FI pups....”

EPA Response: The text was modified as recommended by the commenter to improve clarity.

Peer Reviewer 6 Comment: Under the section Murai et al., 1985, in the second paragraph there is 

reference to the European Commission (2008), when I accessed this reference as per the details in 

the reference list, I did not find a document with NOAEL calculations. Also, did the European 

Commission calculate their NOAEL’s using the data from Murai et al., 1985? This is not clear in 

the text. The LOAELs and NOAELs referred to in the text and cited as Murai et al., must be in the 

Japanese section of the reference as they were not in the English abstract, should this be noted in the 

text?

EPA Response: The text was modified to improve clarity. The revised text is below:

““Female Wistar rats were fed HBCD in the diet at concentrations of 0, 0.01, 0.1, or 1% throughout 

gestation (Days 0 – 20) (Murai et al., 1985; Japanese with English abstract). Dams in the high-dose 

group had a statistically significant decrease (8.4%) in food consumption and increase in liver 

weight (13%) in comparison with controls. There were no treatment-related effects on maternal or 

fetal body weight. There were no effects on the number of implants; number of resorbed, dead, or 

live fetuses; body weight of live fetuses; or incidence of external or visceral abnormalities. A few 

skeletal variations were present but were also observed in controls and not considered significant. 

There were no effects on weaning or survival.

The European Commission (European Commission, 2008) used the study’s data to calculate the 

doses to be 0, 7.5, 75, and 750 mg/kg-day (based on the assumption of a mean animal weight of 

200g and food consumption of 15 g/day). They concluded that the offspring NOAEL was 750 

mg/kg-day and the maternal LOAEL was 750 mg/kg-day based on 13% liver weight increase in the 

high dose group."

3. Do you agree or disagree with the selection of the LOAELs/NOAELs (or BMDLs/BMDs) 

for all endpoints?

Peer Reviewer 4 Comment: The reviewer disagrees with some of the selected 

LOAELs/NOAELS. Please see comments below.
36

P. 40-45, about the study by van der Ven et al. (2009): The liver concentrations of HBCDs in F-1, 

shown in Fig.2 of the original article (p. 55) indicate a significant exposure of the control F-1 to 

HBCDs. In the male F-1 livers, the concentrations of HBCDs in the control are comparable to those 

of the 3 low-dose groups, 0.1, 0.3, and 1 mg/kg bw. In addition, these control levels are higher than 

that of the background levels, which can be discerned in the livers of the control rats in an earlier 

study by van der Ven et al. (2006), see Fig. 1 on p. 287 of the earlier publication. The dose-response 

relationships derived from the results of this study (van der Ven et al., 2009), therefore, can be 

skewed by the observations in the control and the low dose groups.

EPA Response: The following text was added to the hazard assessment to clarify this issue:

“The liver concentrations of HBCDs in F1 control rats, however, indicated exposure of F1 control 

rats to HBCD. For example, in the male F1 livers the concentration of HBCD in the control group 

were comparable to those in the three lowest dose groups.”

Peer Reviewer 4 Comment: P. 45-46, on TWA: For developmental studies, because of different 

susceptibility at different stages of the fetal and postnatal development, it is not appropriate to use 

TWA for the developmental toxicity assessment. 

P. 50 and p. 53: It is not appropriate to use averaging doses over multiple 

reproductive/developmental stages for reproductive/developmental toxicity assessment. 

Development effects are almost always stage specific. In the absence of the knowledge in the target 

stage, the lowest dose across the whole study period should be considered as the effective dose. 

EPA Response: EPA recognizes the importance of developmental windows in such studies. EPA 

took a relatively health conservative approach by using the lowest dose from the dose ranges to 

calculate the time-weighted average dose. Additionally, this hazard assessment is consistent with 

EPA’s use of these time-weighted average doses in past assessments of HBCD (EPA’s Design for 

the Environment Flame Retardant Alternatives for Hexabromocyclododecane and EPA’s 

Preliminary Materials for the Integrated Risk Information System Toxicological Review of 

Hexabromocyclododecane)

Peer Reviewer 4 Comment: P.48, Please state specific effects observed in the 100 mg/kg 

treatment group, including effects on liver wt, epididymides wt, and a clear trend of increased 

thyroid follicular cell hypertrophy etc. These effects should not be ignored; they can be closely 

related to the mechanisms of hypothalamus-pituitary-thyroid axis. 

EPA Response: The changes in relative liver weight and epididymides weight at PNW 11 for 

males in the 100 ppm dose group were added to the text and table. The added text is below:

“Relative liver weight was elevated in the 100 ppm dose group males, but changes were not 

observed at higher doses or in females at any dose level. Relative epididymides weight was also 

elevated in 100 ppm dose group males, but changes were not observed at higher doses.”

The revised table is below:
37

Table 3-3. Summary of effects in parental and F1 rats after dietary, gestational, lactational, 

and postnatal exposure to HBCD

HBCD (ppm in diet)

Malesc Femalesc

Generation 0 100 1,000 10,000 0 100 1,000 10,000

F0

Thyroid

Relative weight (mg/100 g 

BW)

ND ND ND ND 5.73 6.75 6.30 7.47*

Histopathology: diffuse 

follicular cell hypertrophy 

(±/+/++/+++)a

ND ND ND ND 3/10

(0/3/0/0)b

5/10

(2/3/0/0)

6/10

(1/3/2/0)

9/10#

(0/3/4/2)§§

F1

Relative organ weights, PND 20

Liver (g/100 g BW) 3.68 3.82 3.98 4.66* 3.77 3.83 4.01 4.83*

Relative organ weights, PNW 11

Liver (g/100 g BW) 3.45 3.81** 3.58 3.53 3.35 3.59 3.44 3.30

Thyroid (mg/100 g BW) 4.85 5.66 5.78* 6.20** 8.20 6.84 7.35 7.72

Epididymides (mg/100 g 

BW)

0.23 0.21* 0.22 0.21 NA NA NA NA

Thyroid-related hormones

PND 20

T3 (ng/ml) 1.09 1.13 1.06 0.93** ND ND ND ND

T4 (μg/dl) 4.39 4.20 4.78 4.20 ND ND ND ND

TSH (ng/ml) 5.40 6.66 6.07 7.00* ND ND ND ND

PNW 11

T3 (ng/ml) 0.96 0.93 0.88* 0.89** ND ND ND ND

T4 (μg/dl) 4.77 4.84 5.21 5.20 ND ND ND ND

TSH (ng/ml) 4.74 5.81 5.36 4.96 ND ND ND ND

Histopathology

PND 20

Liver: Vacuolar 

degeneration, liver cells, 

diffuse (+/++)a

0/10 0/10 0/10 6/10* 

(6/0)

0/10 0/10 0/10 6/10*

(0/6)§§

PNW 11

Adrenal: Vacuolar 

degeneration, diffuse, 

cortical cells (+/++)a

0/10 0/10 0/10 4/10*

(2/2)§

ND ND ND ND
38

Abbreviations: BW, body weight; PND, postnatal day; ppm, parts per million; PNW, postnatal week; ND, no data; 

NA, not applicable

aGrade of change: (±) minimal; (+) slight; (++) moderate; (+++) severe

bNumber of animals with each grade

cn=10 rats/sex/group

* Significantly different from the controls by Dunnett’s test or Dunnett-type rank-sum test (p<0.05)

** Significantly different from the controls by Dunnett’s test or Dunnett-type rank-sum test (p<0.01)

# Significantly different from the controls by Fisher’s exact probability test (p<0.05)

§ Significantly different from the controls by Mann-Whitney’s U-test (p<0.05)

§§ Significantly different from the controls by Mann-Whitney’s U-test (p<0.01)

Source: Saegusa et al. (2009)

Peer Reviewer 4 Comment: P. 48, “... a LOAEL ... could not be determined...” Please restate a 

conclusion. Only when an entire study showed no effects at all levels one can conclude that no 

LOAEL could be determined. In the case of Saegusa et al. (2009), 8.1 mg/kg is the LOAEL. 

EPA Response: No changes in the observed reproductive parameters were reported (e.g., the 

number of implantation sites, number of live offspring, sex ratio). As such, a reproductive LOAEL 

could not be determined. 

Peer Reviewer 4 Comment: P. 49, Table 3-3: Please consider adding epididymides wt in the table.

EPA Response: Epididymides weight was added to the table. See response above for the revised 

table. 

Peer Reviewer 4 Comment: P. 50, Line 8: please make corrections: Change “measured” to 

“calculated”.

EPA Response: The text was modified to address this comment.

Peer Reviewer 4 Comment: P. 51-53, The significant increases in brain, pituitary gland, and testis 

weights in F1 adults (Table 6 and Table 7 in Ema et al., 2008) should not be dismissed; these 

observations are consistent with the developmental effects of maternal thyroid hormone disturbance 

in the offspring. The study used an acceptable experimental design for reproductive/developmental 

studies. The statistical analysis is appropriate for the study design. 

P. 53: The reviewer suggests a developmental LOAEL of 150 ppm, based on the observed effects 

on testis, brain, and pituitary wts in male F-1 (Tables 6 and 7 of the publication by Ema et al. 

(2008)).

EPA Response: The following text was added to the assessment:

“A significant increase in relative brain weight and relative pituitary gland weight was observed in 

F1 adults at 150 ppm, but there were no significant effects observed at higher doses. Also, a 

significant decrease in absolute brain weight was observed in male and female weanlings and adults 

in the 15,000 ppm dose group. While a significant increase in relative testis weight was observed in 

F1 male weanlings at 150, 1,500, and 15,000 ppm, there were no significant effects on either 
39

absolute or relative testis weights in F1 adults at any dose level. Additionally, these effects were not 

observed in F2 male weanlings. The authors concluded that these organ weight changes were not 

toxicologically significant because they were inconsistent across age, sex, and generation.”

These findings suggest that a LOAEL of 150 ppm based on brain, pituitary, and testis weight 

changes observed in Fl males would not be appropriate. While there was a significant increase in 

relative testis weight in F1 weanlings at 150 ppm, the percent change was relatively small (approx. 

8.7%) and neither absolute nor relative testis weights were significantly changed at any dose group 

in F1 adults. In fact, relative testis weights in F1 adults were slightly, but not significantly, 

decreased compared with the control group. While relative brain weight and relative pituitary gland 

weight were significantly increased in F1 adults at 150 ppm, relative weights were not significantly 

increased in the higher dose groups (1,500 ppm and 15,000 ppm). Due to the lack of a clear doseresponse relationship, EPA did not conclude that 150 ppm should be the LOAEL based on these 

endpoints. 

Peer Reviewer 5 Comment: I agree with the selection of the LOAELs/NOAELs, BMDLs and 

BMDs for all endpoints, as discussed in individual study descriptions. I believe the BMDLs and 

BMDs provide a much more accurate description of effect and non-effect doses than the LOAELS 

and NOAELs, as described in the last section. These values should be given preference in 

describing human health hazards.

EPA Response: Commenter agrees with EPA, no response required.

Peer Reviewer 6 Comment: Please note, mention of LOAELs and NOAELs in other sections. 

Based on the source documents, the LOAELs/NOAELs are accurate, however more clarity 

regarding the EPA calculations would be helpful. Is it possible to have a table with all 

LOAELs/NOAELs reported in the text, all together? My work mostly focuses on human 

biomonitoring of brominated flame retardants and commenting further on LOAELs/NOAELs is 

beyond my expertise.

EPA Response: EPA included which specific endpoint each LOAEL was based on for every 

study. EPA also noted when its LOAELs differed from that of the study authors and included a 

discussion of why those values were chosen. Additionally, EPA included the most relevant 

LOAELs and NOAELs in Figure 3-1. EPA determined that adding a table of LOAELs and NOALs 

would be unnecessarily repetitive with the information already displayed in Figure 3-1. 

4. Are you aware of any additional key published studies or publicly available scientific 

reports that are missing from the draft document that might be useful for discussion of the 

hazards? If so, please provide a copy of the study or a complete citation.

Peer Reviewer 4 Comment: Additional publications are cited within the Comments. The complete 

citation is:

Abdallah MA-E, Harrad S. 2011. Tetrabromobisphenol-A, hexabromocyclododecane and its 

degradation products in UK human milk: Relationship to external exposure. Environment 

International 37(2): 443-448.
40

Brandsma SH, Van der Ven LTM, De Boer J, Leonards PEG. 2009. Identification of hydroxylated 

metabolites of hexabromocyclododecane in wildlife and 28-days exposed Wistar rats. 

Environmental Science & Technology 43(15): 6058-6063. 

Christen V, Crettaz P, Oberli-Schrammli A, Fent K. 2010. Some flame retardants and the 

antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro. Chemosphere 

81(10): 1245-1252.

Hakk H, Szabo DT, Huwe J, Diliberto J, Birnbaum LS. 2012. Novel and distinct metabolites 

identified following a single oral dose of alpha- or gamma-hexabromocyclododecane in mice. 

Environmental Science & Technology 46(24): 13494-13503.

Hinkson NC, Whalen MM. 2010. Hexabromocyclododecane decreases tumor-cell-binding capacity 

and cell-surface protein expression of human natural killer cells. Journal of applied toxicology : 

JAT 30(4): 302-309.

Schecter A, Szabo DT, Miller J, Gent TL, Malik-Bass N, Petersen M, et al., 2012. 

Hexabromocyclododecane (HBCD) stereoisomers in U.S. food from Dallas, Texas. 

Environmental health perspectives 120(9): 1260-1264.

Szabo DT, Diliberto JJ, Huwe JK, Birnbaum LS. 2011a. Differences in tissue distribution of HBCD 

alpha and gamma between adult and developing mice. Toxicological sciences : an official 

journal of the Society of Toxicology 123(1): 256-263.

Szabo DT, Diliberto JJ, Hakk H, Huwe JK, Birnbaum LS. 2011b. Toxicokinetics of the flame 

retardant hexabromocyclododecane alpha: effect of dose, timing, route, repeated exposure, and 

metabolism. Toxicological sciences : an official journal of the Society of Toxicology 121(2): 

234-244.

Szabo DT, Diliberto JJ, Hakk H, Huwe JK, Birnbaum LS. 2010. Toxicokinetics of the flame 

retardant hexabromocyclododecane gamma: effect of dose, timing, route, repeated exposure, 

and metabolism. Toxicological sciences : an official journal of the Society of Toxicology 

117(2): 282-293.

Tomy GT, Budakowski W, Halldorson T, Whittle DM, Keir MJ, Marvin C, et al., 2004. 

Biomagnification of alpha- and gamma-hexabromocyclododecane isomers in a Lake Ontario 

food web. Environmental Science & Technology 38(8): 2298-2303.

Suzuki S, Hasegawa A. 2006. Determination of Hexabromocyclododecane Diastereoisomers and 

Tetrabromobisphenol A in Water and Sediment by Liquid Chromatography/Mass Spectrometry. 

Analytical Sciences 22: 469-474.

EPA Response: Recommended studies were incorporated into the report where appropriate. All 

studies were incorporated except: Christen et al., 2010; Hinkson et al., 2010; Tomy et al., 2004; and 

Suzuki et al., 2006. EPA determined that these studies were not relevant to add to the human health 

hazard assessment because they describe environmental fate and transport (Tomy et al., 2004; 

Suzuki and Hasegawa 2006), in vitro effects on human immune cell binding (Hinkson and Whalen 

2010), and a hypothesized mode of action of HBCD toxicity (Christen et al., 2010). Environmental 

fate and transport and in vitro studies of immune cell binding effects are not relevant for the 

purposes of the human health hazard assessment. EPA did not include the mode of action study 

because EPA cannot speculate on potential modes-of-action for HBCD toxicity in this hazard 

assessment.

Added text for the incorporated studies is below:

“In a more recent study, α-HBCD and γ-HBCD produced distinct metabolites after a single 
41

administration of HBCD to C57BL/6 female mice (Hakk et al., 2012).”

“In a follow-up study, approximately 90% of α-HBCD administrated was absorbed after exposure 

(Szabo et al., 2011b).”

“In a similar study, an administered dose of α-HBCD was found primarily in adipose, liver muscle, 

and skin (Szabo et al., 2011b). Accumulation was dose dependent with nonlinear accumulation at 

higher doses. In a study investigating the differences in tissue distribution of α- and γ-HBCD 

between adult and developing mice, the authors reported that while distribution patterns were 

similar between adult and developing mice, concentrations of each diastereomer were higher in the 

pup’s liver, fat, kidney, brain, blood, muscle, and lungs than the adult’s (Szabo et al., 2011a). This 

finding suggests that developmental stage exposure to α- and γ-HBCD may result in increased body 

burden compared with adults.”

“It is important to note that the composition of HBCD isomers in most rodent toxicity studies 

resembles that of industrial grade HBCD, while human exposure likely contains elevated fraction of 

α-HBCD (Schecter et al., 2012).”

“Abdallah and Harrad (2011) detected two HBCD degradation products for the first time in human 

tissues. The degradation products pentabromocyclododecenes (average concentration of 0.04 ng/g 

lipid weight; n=9) and tetrabromocyclododecadienes (average concentration of 0.15 ng/g lipid 

weight; n=25) were both detected in human milk samples from Birmingham, UK.”

“In another study, four different groups of hydroxylated HBCD metabolites were identified in rat 

adipose, liver, lung, and muscle tissue by liquid and gas chromotography combined with mass 

spectrometry: monohydroxy metabolites of HBCD, pentabromocyclododecene (PBCDe), 

tetrabromocyclododecene (TBCDe), and dihydroxy-HBCD (Brandsma et al., 2009).”

Peer Reviewer 5 Comment: I am not aware of any additional key published studies or publicly 

available scientific reports that are missing from the draft document. I conducted a Medline search 

and did not come up with any additional citations.

EPA Response: No response required.

Peer Reviewer 6 Comment:

Brominated flame retardant concentrations in sera from the Canadian Health Measures Survey 

(CHMS) from 2007 to 2009. Rawn DF, Ryan JJ, Sadler AR, Sun WF, Weber D, Laffey P, Haines 

D, Macey K, Van Oostdam J. Environ Int. 2014.

Tetrabromobisphenol A and hexabromocyclododecane flame retardants in infant-mother paired 

serum samples, and their relationships with thyroid hormones and environmental factors. Kim 

UJ, Oh JE. Environ Pollut. 2014 Jan;184:193-200.

Neurobehavioral function and low-level exposure to brominated flame retardants in adolescents: a 

cross-sectional study. Kiciński M, Viaene MK, Den Hond E, Schoeters G, Covaci A, Dirtu AC, 

Nelen V, Bruckers L, Croes K, Sioen I, Baeyens W, Van Larebeke N, Nawrot TS. Environ 

Health. 2012 Nov 14;11:86.
42

Brominated flame retardants in the Australian population: 1993-2009. Toms LM, Guerra P, Eljarrat 

E, Barceló D, Harden FA, Hobson P, Sjodin A, Ryan E, Mueller JF. Chemosphere. 2012 

Oct;89(4):398-403.

EPA Response: Recommended studies were incorporated into the report where appropriate. All 

studies were incorporated. 

The added text is below:

“In an analysis of HBCD concentrations in sera from the Canadian Health Measures Survey, 

researchers detected HBCD in all samples analyzed (Rawn et al., 2014). In most samples, HBCD 

was detected as <1 ng/g lipid, similar to global concentrations. α-HBCD was the biggest contributor 

to total HBCD, although β-HBCD and γ-HBCD were detected in 23% and 35% of the samples, 

respectively. No differences in total HBCD concentration were associated with age or sex.”

“More recently, significant correlations between HBCD concentrations in sera from mothers and 

infants have been found, suggesting that that maternal transfer is important (Kim and Oh, 2014).”

“No temporal trend, however, was apparent in a study conducted using human breast milk samples 

collected in Australia from 1993 to 2009 (Toms et al., 2012).”

“Kiciński et al (2012) assessed the relationship between PBDE exposure and neurobehavioral 

function using biomonitoring data from Flanders, Belgium. They concluded that PBDE exposure 

was associated with changes in the motor function and serum levels of thyroid hormones, but 

HBCD did not show significant associations with performance in the neurobehavioral tests. These 

authors conclude, however, that the potential for exposure misclassification in this study may have 

been likely due to a large number of observations for which PBDE levels were below the level of 

quantification. As a result, the authors had to create categories that were not biologically relevant in 

order to analyze the data.”

5. Please provide any additional comments or suggestions you may have that may improve 

this section of the document.

Peer Reviewer 4 Comment: P. 29, Last Line, for correction: A dose in the study, 2150 mg/kg, 

needs to be inserted in this sentence. 

EPA Response: The missing dose was added to the sentence. 

Peer Reviewer 4 Comment: Section 3.2.1, Absorption: There are no discussions on dermal or 

inhalation absorption. 

EPA Response: No studies focused on dermal or inhalation absorption were identified in the 

literature.

Peer Reviewer 4 Comment: P. 31 IRDC (1977) Acute Inhalation Toxicity Study in Rats: “339.65 

grams... for 4 hours” does not provide sufficient information to describe the dose. 
43

EPA Response: The text was modified to address this comment. Below is the revised text:

“Male and female Charles River CD rats (5/sex) were placed in cages in stainless steel chamber 

with a constant airflow. A total of 339.65 grams of HBCD dust powder was disseminated in a 4 

hour exposure period. The rate of powder disseminated was calculated to be 1.415 grams per 

minute. The chamber dust concentration was calculated to be 202.14 mg/L. Mortality was not 

observed. All animals demonstrated an increase in body weight and slight dyspnea was observed

during the 4-hour exposure duration, but there was no control group with which to compare the 

treated animals.”

Peer Reviewer 4 Comment: The composition of isomers in most of HBDC tested in the toxicity 

studies resembles that of industrial grade HBCD, while human exposure to environmental and 

dietary contamination, contains elevated fraction of alpha-HBCD (Schecter et al., 2012). AlphaHBCD has much longer half-lives than the beta- and gamma-isomers in human. This contributes to 

an additional uncertainty when applying the results of these studies to assess the toxicity in human

HBCD sterioisomers in food have been examined by Schecter et al. (2012).

EPA Response: The following text was added to Section 3.2.2: 

“It is important to note that the composition of HBCD isomers in most rodent toxicity studies 

resembles that of industrial grade HBCD, while human exposure likely contains elevated fraction of 

α-HBCD (Schecter et al., 2012).”

Peer Reviewer 4 Comment: Here is an article on isomer bioaccumulation in Lake Ontario Food 

Web (Tomy et al., 2004), another one on gama isomer and others in sediment (Suzuki and 

Hasegawa 2006). 

Studies of HBCD effect on human natural killer (NK) cells in vitro demonstrated that HBCD 

decreases tumor-cell-binding and cell-surface protein expression of NK cells (Hinkson and Whalen 

2010), suggesting that HBCD has the potential to increase cancer incidence and viral infections.

EPA Response: EPA determined that these studies were not relevant to add to the human health 

hazard assessment because they describe environmental fate and transport (Tomy et al., 2004; 

Suzuki and Hasegawa 2006) and in vitro effects on human immune cell binding (Hinkson and 

Whalen 2010). Neither of these topics are relevant for the purposes of this human health hazard 

assessment.

Peer Reviewer 4 Comment: Three of Szabo’s articles may be useful, so is the one by Hakk et al. 

(2012): (Szabo et al., 2011a; Szabo et al., 2010, 2011b) (Hakk et al., 2012)

EPA Response: All of these studies were included in the report. The added text is below:

“In a more recent study, α-HBCD and γ-HBCD produced distinct metabolites after a single 

administration of HBCD to C57BL/6 female mice (Hakk et al., 2012).”
44

“In a follow-up study, approximately 90% of α-HBCD administrated was absorbed after exposure 

(Szabo et al., 2011b).”

“In a similar study, an administered dose of α-HBCD was found primarily in adipose, liver muscle, 

and skin (Szabo et al., 2011b). Accumulation was dose dependent with nonlinear accumulation at 

higher doses. In a study investigating the differences in tissue distribution of α- and γ-HBCD 

between adult and developing mice, the authors reported that while distribution patterns were 

similar between adult and developing mice, concentrations of each diastereomer were higher in the 

pup’s liver, fat, kidney, brain, blood, muscle, and lungs than the adult’s (Szabo et al., 2011a). This 

finding suggests that developmental stage exposure to α- and γ-HBCD may result in increased body 

burden compared with adults.”

Peer Reviewer 5 Comment: A confusing element of the EPA report and of the literature is that 

very different effect/no-effect doses have been obtained from more traditional studies using 

NOAELs and LOAELs than more recent studies using BDMLs and BMDs, with the latter yielding 

far lower effect doses than the former. In my opinion, analysis of dose-response data using the 

BMDL approach is more scientifically valid and relevant to human health assessment than the 

NOAEL/LOAEL approach. A priori identification of a critical effect size (CES) from which the 

critical effect dose (CED) is identified is a more scientifically valid approach than routine 

evaluation. Use of the 5% lower confidence limit of the CED can then be used to identify the 

benchmark loses lower bound confidence limit (BMDL). It would be beneficial if there was a 

discussion of this issue at the end of the Summary Section of the Human Health Hazard section, as 

well as that this is the preferred approach taken by the EPA. Otherwise, it looks as if there is a 

collection of contradictory studies from which no conclusions can be drawn.

EPA Response: It is beyond the scope of this hazard assessment to discuss the methodological 

considerations of using a NOAEL/LOAEL approach versus a BMD/BMDL approach. A detailed 

explanation of the BMD/BMDL approach (including CES and CED) used in the Saegusa et al. 

(2009) study was included in the assessment. 

Peer Reviewer 6 Comment: The document reads very well, but please see within my answers to 

the questions above, some suggestions to improve the document. Is this review intended to be a 

review of “all” the literature on HBCD and health effects or rather a selection of literature. Could 

this (and perhaps an aim) be specified in the beginning of the document?

EPA Response: The assessment is an attempt to summarize the most relevant literature regarding 

the human health hazard for HBCD to assess whether the potential human health effects would 

support listing under the EPCRA Section 313 criteria. EPA incorporated all of the relevant 

publications suggested by all of the peer reviewers. 
45

Response to External Peer Review Section III: Ecological Hazard Assessment

I. Charge to Reviewers

1. Does the summary statement and review of the literature present a clear rationale and 

accurately reflect the conclusions regarding the ecological hazards of HBCD?

2. Please comment on the characterization of HBCD’s potential for bioaccumulation. Has the 

scientific evidence been accurately and clearly represented and evaluated? Do you agree or 

disagree with the conclusions?

3. Are there additional key published studies or publicly available scientific reports that are 

missing from the draft document that might be useful for discussion of the ecological 

hazards?

4. Please provide any additional comments or suggestions you may have that may improve this 

section of the document.

II. Response to Ecological Hazard Assessment Charge 

A. General Impressions

None were provided by the peer reviewers.

B. Peer Reviewer Comments by Charge Question

1. Does the summary statement and review of the literature present a clear rationale and 

accurately reflect the conclusions regarding the ecological hazards of HBCD?

Peer Reviewer 7 Comment: The rationale for the assessment seems fairly clear based on the 

bioaccumulation potential and toxicity data presented in the literature review. The summary does 

reflect accurately the conclusions regarding the ecological hazards of HBCD with one exception 

related to the thyroid axis. Overall the summary adequately captures the major points of the 

literature review but there are several issues that should be clarified. 

First, sub lethal effects on thyroid axis have been reported in rainbow trout as summarized in the 

third paragraph on page 86. Given the fact that the thyroid gland seems to be targeted by HBCD in 

mammalian studies, and the fact that many aspects of thyroid physiology are evolutionarily 

conserved, I would have expected some aspect of the thyroid effects to be mentioned in the 

summary. I would be happy to provide some review papers in support of the evolutionary 

conservation of thyroid function if needed. 

EPA Response: Added expanded information and discussion, including additional citations, on 

thyroid effects in section 4.1 to include more detailed discussion of effects in fish, birds, and 

mammals. Added text: 

“A wide range of effects of HBCD have been reported in fish (e.g., developmental toxicity, embryo 

malformations, reduced hatching success, reduced growth, hepatic enzyme and biomarker effects, 

thyroid effects, DNA damage to erythrocytes, and oxidative damage) and invertebrates (e.g., 

degenerative changes, morphological abnormalities, decreased hatching success, and altered 
46

enzyme activity) (Anselmo et al., 2011; Deng et al., 2009; Du et al., 2012a; Hong et al., 2014a; Hu 

et al., 2009; Palace et al., 2008, 2010; Ronisz et al., 2004; Smolarz and Berger, 2009; Wu et al., 

2013; Zhang et al., 2008, 2014a). Effects on the thyroid in fish (reduced thyroid hormone 

(triiodothyronine, T3, and thyroxine, T4) levels in rainbow trout; Palace et al., 2008, 2010), are 

similar to those observed in mammals (see Chapter 3, Human Health Hazard Assessment). Reduced 

T4 levels were also reported in birds exposed to HBCD (Marteinson et al., 2011).”

“Thyroid effects were reported in juvenile rainbow trout (Oncorhynchus mykiss) following dietary 

exposure to HBCD (Palace et al., 2008, 2010). Each of the diastereoisomers of HBCD 

(administered separately via diet at concentrations of 5 ng/g of α-, β-, or γ-HBCD for up to 56 days) 

disrupted thyroid homeostasis, as indicated by lower free circulating T3 and T4 levels (Palace et al., 

2008, 2010).”

Peer Reviewer 7 Comment: Secondly, the summary and literature review are missing a clear 

indication of which criteria were used in selecting the studies discussed. Were only studies that 

followed EPA technical guidelines assessed in the Ecological Risk Assessment section? Were 

studies that employed flow through conditions weighted more heavily than static tests? Many of the 

newer references are absent from the literature review, so was there a cutoff used for the literature 

searches. While I do tend to agree with the conclusion that "HBCD is expected to present a concern 

for adverse effects on the environment" it isn't entirely clear what criteria were used to select the 

studies presented in Table 4.1. Moreover it is unclear if more weight was given to studies that 

followed EPA guidelines or that used flow through conditions, for example. It seems that the 

summary should include the criteria for which papers were used to reach their conclusion, aside 

from just the databases which were searched (which appears in 4.2). If all papers were given equal 

weight at this point in the assessment than state such. For example, on page 69, a long quote from 

the OECD is used related to the testing of difficult substances due to their low solubility in water. 

We are given the impression that the authors have relied on this in some way (why else does it 

appear here?) but there is no mention in the summary of the extent to which the OECD guidelines 

were used and studies were considered invalid on a case by case basis. In at least one case the 

authors report (p 77, Table 4.1) an 'invalid' study in their analysis, but there is no indication in the 

summary or in 4.2 how this was determined. Again, if in this case OECD criteria were used then it 

would be helpful to include this in the discussion.

EPA Response: Expanded discussion in section 4.2 to include more detail on quality criteria and 

guidelines for study selection. In addition, references were updated to include studies up through 

2015. Revised discussion paragraphs:

“This assessment includes not only studies that followed standard test guidelines 

(e.g., Office of Chemical Safety and Pollution Prevention [OCSPP], Organisation for Economic Cooperation and Development [OECD]), but also non-standard toxicity tests that followed procedures 

that were scientifically sound. Only clearly adverse signs of toxicity (e.g., lethality, immobility, 

effects on growth and reproduction, organ histopathology, abnormal behavior) were used to set 

toxicity effect levels such as no-observed-effect concentrations (NOECs) and lowest-observedeffect concentrations (LOECs). Although studies in which the nominal concentrations greatly 

exceeded water solubility (e.g., Siebel-Sauer and Bias, 1987 as cited in ECB, 2008; Jatzek, 1990; 

Calmbacher, 1978) are described in this assessment, the conclusion of concern for adverse effects 

on the environment is not based on these studies, as none of these studies reported toxic effects. 
47

Studies were considered to be invalid if the control response was abnormal.

Commercial mixtures of HBCD, or technical HBCD (HBCD-TM), consist primarily of three 

diastereomers: α-HBCD, β-HBCD, and γ-HBCD. Each of these diastereomers has two enantiomers 

(+ and ̶ ); the ratio between these two enantiomers is often reported as the enantiomeric fraction 

(EF). Enantiomers of chiral compounds such as HBCD often exhibit differences in biological 

activity (Zhang et al., 2009). As summarized in de Wit et al. (2010), γ-HBCD tends to predominate 

in air, α-HBCD is the most abundant diastereomer in biota, and α-HBCD, β-HBCD, and γ-HBCD 

have been found in relatively similar proportions in marine sediments. More recent environmental 

monitoring studies are reporting concentrations of individual diastereomers (e.g., Chen et al., 2011) 

and ecotoxicological studies are reporting acute and chronic effects of individual diastereomers of 

HBCD (e.g., Du et al., 2012b). 

HBCD is poorly water soluble and tends to partition to organic phases in the aquatic 

environment (e.g., lipids, suspended solids; see Marvin et al., 2011). Assessment of aquatic toxicity 

is complicated by the low water solubility of the chemical and differences in the solubility of the 

three main HBCD isomers, which makes testing difficult and interpretation uncertain for studies 

conducted above the water solubility. Aquatic studies available to assess ecotoxicity concerns 

generally used solvents such as dimethyl sulfoxide (DMSO) to solubilize HBCD into solution and 

generally did not characterize the impact that solvents had on test solutions. According to OECD 

guidance for testing difficult substances (OECD, 2000), “because of the potential for interaction 

with the test substance resulting in an altered response in the test, their use should be restricted to 

situations where no other acceptable method of media preparation is available.” If use of a solvent is 

deemed necessary, OECD (2000) recommends that a preliminary experiment be conducted that 

determines solubility in test solutions under testing conditions. OECD (2000) includes a caveat that 

“a high level of uncertainty is often associated with solubility estimates for poorly water-soluble 

substances. It is therefore often not possible to specify an upper exposure limit with a high degree of 

confidence. Exposure concentrations which exceed a “reported” water solubility should therefore 

not necessarily be considered invalid but reviewed on a case-by-case basis.” Studies in which an 

organic solvent was used were included in this assessment; the use of a solvent is noted in the study 

summary. For studies in which nominal concentrations greatly exceeded the water solubility of 

HBCD, a comment stating such has been included in the study description. It should be noted that 

water solubility studies available for HBCD indicate differences among the water solubilities of the 

three diastereomers, α-, β-, and γ-HBCD (0.0488, 0.0147, and 0.0021 – 0.0034 mg/L for α-, β-, and 

γ-HBCD, respectively), which suggests that overall water solubility of technical-grade HBCD may 

vary depending on its isomeric composition. Also, a solvent may differentially alter the solubility of 

each of the HBCD diastereomers. Presently, insufficient information is available to address whether 

use of solvents affects the outcomes of available studies or to establish a more definitive limit of 

water solubility.”

The following references have been added to the assessment:

Andersen, MS; Fuglei, E; König, M; et al. (2015) Levels and temporal trends of persistent organic 

pollutants (POPs) in arctic foxes (Vulpes lagopus) from Svalbard in relation to dietary habits and 

food availability. Sci. Total. Environ. 511:112–122.

Anselmo, HMR; Koerting, L; Devito, S; et al. (2011) Early life developmental effects of marine 
48

persistent organic pollutants on the sea urchin Psammechinus miliaris. Ecotox. Environ. Safe.

74:2182–2192.

Brandsma, SH; Leonards, PEG; Leslie, HA; et al. (2015) Tracing organophosphorus and 

brominated flame retardants and plasticizers in an estuarine food web. Sci. Total. Environ. 505:22–

31.

Bustnes, JO; Borgå, K; Dempster, T; et al. (2012) Latitudinal distribution of persistent organic 

pollutants in pelagic and demersal marine fish on the Norwegian coast. Environ. Sci. Technol.

46:7836–7843.

Cheaib, Z; Grandjean, D; Kupper, T; et al. (2009) Brominated flame retardants in fish of Lake 

Geneva (Switzerland). Bull. Environ. Contam. Toxicol. 82:522–527.

Chen, D; La Guardia, MJ; Luellen, DR; et al. (2011) Do temporal and geographical patterns of 

HBCD and PBDE flame retardants in U.S. fish reflect evolving industrial usage? Environ. Sci.

Technol. 45:8254–8261.

Chen, D; Letcher, RJ; Burgess, NM; et al. (2012) Flame retardants in eggs of four gull species 

(Laridae) from breeding sites spanning Atlantic to Pacific Canada. Environ. Pollut. 168:1–9.

Crump, D; Egloff, C; Chiu, S; et al. (2010) Pipping success, isomer-specific accumulation, and 

hepatic mRNA expression in chicken embryos exposed to HBCD. Toxicol. Sci. 115:492–500.

de Wit, CA; Herzke, D; Vorkamp, K. (2010) Brominated flame retardants in the Arctic environment 

– trends and new candidates. Sci Total Environ 408:2885–2918.

Dodder, NG; Maruya, KA; Ferguson, PL; et al. (2014) Occurrence of contaminants of emerging 

concern in mussels (Mytilus spp.) along the California coast and the influence of land use, storm 

water discharge, and treated wastewater effluent. Marine Poll. Bull. 81:340–346.

Du, M; Lin, L; Yan, C; et al. (2012a) Diastereoisomer- and enantiomer-specific accumulation, 

depuration, and bioisomerization of hexabromocyclododecanes in zebrafish (Danio rerio). Environ.

Sci. Technol. 46:11040–11046.

Du, M; Zhang, D; Yan, C; et al. (2012b) Developmental toxicity evaluation of three 

hexabromocyclododecane diastereoisomers on zebrafish embryos. Aquat. Toxicol. 112–113:1–10.

Du, M; Lin, L; Yan, C; et al. (2013) Enantiomer-specific bioaccumulation and depuration of 

hexabromocyclododecanes in zebrafish (Dania rerio). J. Haz. Mater. 248–249:167–171.

ECOSAR. (2012) ECOSAR: v. 1.11. U.S. Environmental Protection Agency, OPPT-Risk 

Assessment Division. Available online at 

http://www.epa.gov/opptintr/newchems/tools/21ecosar.htm (accessed June 17, 2015).

Esslinger, S; Becker, R; Müller-Belecke, A; et al. (2010) HBCD sereoisomer pattern in mirror carps 

following dietary exposure to pure γ-HBCD enantiomers. J. Agric. Food Chem. 58:9705–9710.
49

Eulaers, I; Jaspers, VLB; Pinxten, R; et al. (2014) Legacy and current-use brominated flame 

retardants in the barn owl. Sci. Total Environ. 472:454–462.

Fernie, KJ; Letcher, RJ. (2010) Historical contaminants, flame retardants, and halogenated phenolic 

compounds in peregrine falcon (Falco peregrinus) nestlings in the Canadian Great Lakes basin. 

Environ. Sci. Technol. 44:3520–3526.

Fernie, KJ; Marteinson, SC; Bird, DM; et al. (2011) Reproductive changes in American kestrels 

(Falco sparverius) in relation to exposure to technical hexabromocyclododecane flame retardant. 

Environ. Toxicol. Chem. 30:2570–2575.

Foekema, EM; Parron, ML; Mergia, MT; et al. (2014) Internal effect concentrations of organic 

substances for early life development of egg-exposed fish. Ecotox. Environ. Safe. 101:14–22.

Guerra, P; Alaee, M; Jiménez, B; et al. (2012) Emerging and historical brominated flame retardants 

in peregrine falcon (Falco peregrinus) eggs from Canada and Spain. Environ. Int. 40:179–186.

Haukås, M; Hylland, K; Nygård, T; et al. (2010a) Diastereomer-specific bioaccumulation of 

hexabromocyclododecane (HBCD) in a coastal food web, Western Norway. Sci. Total Environ.

408:5910–5916.

Haukås, M; Ruus, A; Hylland, K; et al. (2010b) Bioavailability of hexabromocyclododecane to the 

polychaete Hediste diversicolor: exposure through sediment and food from a contaminated fjord. 

Environ. Toxicol. Chem. 29:1709–1715.

He, M; Luo, X; Yu, L; et al. (2013) Diasteroisomer and enantiomer-specific profiles of 

hexabromocyclododecane and tetrabromobisphenol A in an aquatic environment in a highly 

industrialized area, South China: vertical profile, phase partition, and bioaccumulation. Environ.

Poll. 179:105–110.

Hong, H; Li, D; Shen, R; et al. (2014a) Mechanisms of hexabromocyclododecanes induced 

developmental toxicity in marine medaka (Oryzias melastigma) embryos. Aquat. Toxicol. 152:173–

185.

Hong, SH; Shim, WJ; Han, GM; et al. (2014b) Levels and profiles of persistent organic pollutants 

in resident and migratory birds from an urbanized coastal region of South Korea. Sci. Total 

Environ. 470–471:1463–1470.

Hu, J; Liang, Y; Chen, M; et al. (2009) Assessing the toxicity of TBBPA and HBCD by zebrafish 

embryo toxicity assay and biomarker analysis. Environ. Toxicol. 24:334–342.

Hu, J; Jim, J; Wang, Y; et al. (2011) Levels of polybrominated diphenyl ethers and 

hexabromocyclododecane in the atmosphere and tree back from Beijing, China. Chemosphere 

84:355–360.

Ilyas, M; Sudaryanto, A; Setiawan, IE; et al. (2013) Characterization of polychlorinated biphenyls 
50

and brominated flame retardants in sludge, sediment and fish from municipal dumpsite at Surabaya, 

Indonesia. Chemosphere 93:1500–1510.

Isobe, T; Ochi, Y; Ramu, K; et al. (2009) Organohalogen contaminants in striped dolphins (Stenella 

coeruleoalba) from Japan: present contamination status, body distribution and temporal trends 

(1978–2003). Marine Poll. Bull. 58:396–401.

Isobe, T; Ogawa, SP; Ramu, K; et al. (2012) Geographical distribution of non-PBDE-brominated 

flame retardants in mussels from Asian coastal waters. Environ. Sci. Pollut. Res. 19:3107–3117.

Jeong, GH; Hwang, NR; Hwang, E; et al. (2014) Hexabromocyclododecanes in crucian carp and 

sediment from the major rivers in Korea. Sci. Total. Environ. 470–471:1471–1478.

Jin, L; Lam, JCW; Murphy, MB; et al. (2012) Temporal trends of polybrominated diphenyl ethers 

and hexabromocyclododecanes in marine mammals with special reference to Hong Kong, South 

China. In: Loganathan, BG; Lam, PKS, eds. Global Contamination Trends of Persistent Organic 

Chemicals. Boca Raton, FL: CRC Press, pp 497–513.

Klosterhaus, SL; Stapleton, HM; La Guardia, MJ; et al. (2012) Brominated and chlorinated flame 

retardants in San Francisco Bay sediments and wildlife. Environ. Int. 47:56–65.

La Guardia, MJ; Hale, RC; Harvey, E; et al. (2012) In situ accumulation of HBCD, PBDEs, and 

several alternative flame-retardants in the bivalve (Corbicula fluminea) and gastropod (Elimia 

proxima). Environ. Sci. Technol. 46:5798–5805.

Law, RJ; Covaci, A; Harrad, S; et al. (2014) Levels and trends of PBDEs and HBCDs in the global 

environment: status at the end of 2012. Environ. Int. 65:147–158.

Letcher, RJ; Gebbink, WA; Sonne, C; et al. (2009) Bioaccumulation and biotransformation of 

brominated and chlorinated contaminants and their metabolites in ringed seals (Pusa hispida) and 

polar bears (Ursus maritimus) from East Greenland. Environ. Int. 35:1118–1124.

Li, Y; Zhou, Q; Wang, Y; et al. (2011) Fate of tetrabromobisphenol A and 

hexabromocyclododecane brominated flame retardants in soil and uptake by plants. Chemosphere 

82:204–209.

Malarvannan, G; Belpaire, C; Geeraerts, C; et al. (2014) Assessment of persistent brominated and 

chlorinated organic contaminants in the European eel (Anguilla anguilla) in Flanders, Belgium: 

Levels, profiles, and health risk. Sci. Total Environ. 482–483:222–233. 

Marteinson, SC; Kimmins, S; Letcher, RJ; et al. (2011) Diet exposure to technical 

hexabromocyclododecane (HBCD) affects testes and circulating testosterone and thyroxine levels in 

American kestrels (Falco sparverius). Environ. Res. 111:1116–1123.

Marteinson, SC; Bird, DM; Letcher, RJ; et al. (2012) Dietary exposure to technical 

hexabromocyclododecane (HBCD) alters courtship, incubation and parental behaviors in American 

kestrels (Falco sparverius). Chemosphere 89:1077–1083.
51

Marvin, CH; Tomy, GT; Armitage, JM; et al. (2011) Hexabromocyclododecane: current 

understanding of chemistry, environmental fate and toxicology and implications for global 

management. Environ. Sci. Technol. 45:8613–8623.

McKinney, MA; Letcher, RJ; Aars, J; et al. (2011) Flame retardants and legacy contaminants in 

polar bears from Alaska, Canada, East Greenland and Svalbard, 2005–2008. Environ. Int. 37:365–

374.

Miller, A; Elliott, JE; Elliott, KH; et al. (2015) Brominated flame retardant trends in aquatic birds 

from the Salish Sea region of the west coast of North America, including a mini-review of recent 

trends in marine and estuarine birds. Sci. Total Environ. 502:60–69.

Ronisz, D; Farmen Finne, E; Karlsson, H; et al. (2004) Effects of the brominated flame retardants 

hexabromocyclododecane (HBCDD), and tetrabromobisphenol A (TBBPA), on hepatic enzymes 

and other biomarkers in juvenile rainbow trout and feral eelpout. Aquat. Toxicol. 69:229–245.

Salamova, A; Hites, RA. (2013) Brominated and chlorinated flame retardants in tree bark from 

around the globe. Environ. Sci. Technol. 47:349–354.

Sellström, U; Kierkegaard, A; de Wit, C; et al. (1998) Polybrominated diphenyl ethers and 

hexabromocyclododecane in sediment and fish from a Swedish river. Environ. Toxicol. Chem.

17:1065–1072.

Shaw, SD; Berger, ML; Weijs, L; et al. (2012) Tissue-specific accumulation of polybrominated 

diphenyl ethers (PBDEs) including deca-BDE and hexabromocyclododecanes (HBCDs) in harbor 

seals from the northwest Atlantic. Environ. Int. 44:1–6.

Sun, Y; Luo, X; Mo, L; et al. (2012) Hexabromocyclododecane in terrestrial passerine birds from ewaste, urban and rural locations in the Pearl River Delta, South China: levels, biomagnification, 

diastereoisomer- and enantiomer-specific accumulation. Environ. Pollut. 171:191–198.

Venier, M; Wierda, M; Bowerman, WW; et al. (2010) Flame retardants and organochlorine 

pollutants in bald eagle plasma from the Great Lakes region. Chemosphere 80:1234–1240.

Vorkamp, K; Rig, FF; Bossi, R; et al. (2011) Temporal trends of hexabromocyclododecane, 

polybrominated diphenyl ethers and polychlorinated biphenyls in ringed seals from East Greenland. 

Environ. Sci. Technol. 45:1243–1249.

Watanabe, I; Tatsukawa, R. (1989) Anthropogenic brominated aromatics in the Japanese 

environment. Proceedings of the Workshop on Brominated Aromatic Flame Retardants, Swedish 

National Chemicals Inspectorate, Solna, Sweden, October 24–26, pp 63–71 [as cited in Sellström et 

al. 1998].

Wu, J; Guan, Y; Zhang, Y; et al. (2010) Trophodynamics of hexabromocyclododecanes and several 

other non-PBDE brominated flame retardants in a freshwater food web. Environ. Sci. Technol.

44:5490–5495.
52

Wu, J; Guan, Y; Zhang, Y; et al. (2011) Several current-use, non-PBDE brominated flame 

retardants are highly bioaccumulative: evidence from field determined bioaccumulation factors. 

Environ. Int. 37:210–215.

Wu, J; Zhang, Y; Luo, X; et al. (2012a) A review of polybrominated diphenyl ethers and alternative 

brominated flame retardants in wildlife from China: levels, trends, and bioaccumulation 

characteristics. J. Environ. Sci. 24:183–194.

Wu, T; Wang, S; Huang, H; et al. (2012b) Diastereomer-specific uptake, translocation, and toxicity 

of hexabromocyclododecane diastereoisomers to maize. J. Agr. Food Chem. 60:8528–8534.

Wu, M; Zuo, Z; Li, B; et al. (2013) Effects of low-level hexabromocyclododecane (HBCD) 

exposure on cardiac development in zebrafish embryos. Ecotoxicology 22:1200–1207.

Yu, L; Luo, X; Zheng, X; et al. (2013) Occurrence and biomagnification of organohalogen 

pollutants in two terrestrial predatory food chains. Chemosphere 93:506–511.

Zhang, Y; Yang, F; Luo, C; et al. (2009) Bioaccumulative characteristics of 

hexabromocyclododecanes in freshwater species from an electronic waste recycling area in China. 

Chemosphere 76:1572–1578.

Zhang, Y; Sun, H; Liu, F; et al. (2013) Hexabromocyclododocanes in limnic and marine organisms 

and terrestrial plants from Tiangin, China: diastereomer- and enantiomer-specific profiles, 

biomagnification, and human exposure. Chemosphere 93:1561–1568.

Zhang, H; Pan, L; Tao, Y. (2014a) Antioxidant responses in clam Venerupis philippinarum exposed 

to environmental pollutant hexabromocyclododecane. Environ. Sci. Pollut. Res. 21:8206–8215.

Zhang, Y; Sun, H; Ruan, Y. (2014b) Enantiomer-specific accumulation, depuration, metabolization 

and isomerization of hexabromocyclododecane (HBCD) diastereomers in mirror carp from water. J.

Haz. Mater. 264:8–15.

Zhang, Y; Sun, H; Zhu, H; et al. (2014c) Accumulation of hexabromocyclododecane diastereomers 

and enantiomers in two microalgae, Spirulina subsalsa and Scenedesmus obliquus. Ecotox. Environ.

Safe. 104:136–142.

Zhu, N; Fu, J; Gao, Y; et al. (2013) Hexabromocyclododecane in alpine fish from the Tibetan 

Plateau, China. Environ. Poll. 181:7–13.

Peer Reviewer 7 Comment: Third, an editorial point- the BCF and BMF values presented in the 

third sentence of the summary appear also in the last paragraph of the summary on page 68. This 

seems redundant and detracts from the clarity of the summary. 

EPA Response: Removed redundancy in discussion of BCF and BMF in section 4.1.
53

Peer Reviewer 8 Comment: The summary statement and review of the literature presents a clear 

rationale and accurately reflect the conclusions regarding the ecological hazards of HBCD. To 

summarize, HBCD has significant propensity for bioaccumulation and biomagnification. Due to the 

extremely low water solubility HBCD, water borne exposures for toxicity testing may be highly 

uncertain because of the necessity to utilize carrier solvents. The predicted toxicity appears relevant 

to other receptors and should suffice for uncertainties within the water column and the accumulative 

nature of the compounds.

EPA Response: Commenter agrees with EPA’s assessment, no action required or taken based on 

this comment.

Peer Reviewer 9 Comment: The report provides a clear statement on the ecological hazards for 

HBCD. This includes specific comments on bioaccumulation, and biomagnification and evidence of 

toxicity to aquatic and terrestrial species including plants, invertebrates and vertebrates. The report 

specifically addresses the concern that HBCD is not highly soluble in water which can confound the 

assessment of toxicity in aquatic species.

EPA Response: Commenter agrees with EPA’s assessment No action required or taken based on 

this comment.

2. Please comment on the characterization of HBCD’s potential for bioaccumulation. Has the 

scientific evidence been accurately and clearly represented and evaluated? Do you agree or 

disagree with the conclusions? 

Peer Reviewer 7 Comment: The older literature on HBCD accumulation and depuration has been 

accurately and clearly presented and I agree with the conclusions of the authors. However more 

recent 2014 studies (below) should be included and the literature and summary updated 

accordingly.

EPA Response: The suggested studies were reviewed and a new literature search was performed. 

Updated discussion and additional references were added where appropriate.

Introductory text for bioaccumulation was expanded, clarified and extensively rewritten (including 

updated references):

“Bioaccumulation in aquatic organisms can be estimated using parameters such as BCFs, 

bioaccumulation factors (BAFs), biota-sediment accumulation factors (BSAFs), BMFs, trophic 

magnification factors (TMFs), and assimilation efficiencies. BCFs, BAFs, BSAFs, and assimilation 

efficiencies describe the degree to which a chemical can be concentrated in the tissues of an 

organism. BCFs account for exposure from the ambient environment only (typically water) through 

an organism’s respiratory and dermal surfaces; BAFs account for absorption from all routes of 

exposure, including diet, in the natural environment (Arnot and Gobas, 2006); BSAFs are BAFs for 

organisms living in sediment; and assimilation efficiency represents an estimate of the fraction of 

the BAF that may be accumulated from the diet. A BCF, BAF, or BSAF greater than 1 implies 

bioaccumulation from the environment. 

A BMF expresses the accumulation of a contaminant in a given species from the species in the 

trophic level below it and is calculated as the concentration of a contaminant in the organism 
54

divided by the concentration of the contaminant in its prey; this is similar to a TMF, which is the 

average rate of increase of a contaminant from one trophic level to the next in a food web. A BMF 

or TMF greater than 1 indicates potential for biomagnification through the food chain, such that a 

predator species would have higher body burdens of the chemical than its prey species. 

HBCD has been shown in numerous studies to bioaccumulate and biomagnify in aquatic and 

terrestrial food chains. In general, α-HBCD bioaccumulates in organisms and biomagnifies through 

food webs to a greater extent than the β- and γ- diastereoisomers. Uncertainty remains as to the 

balance of diastereoisomer accumulation in various species and the extent to which 

bioisomerization and biotransformation rates for each isomer affect bioaccumulation potential. 

Some authors (e.g., Law et al., 2006) have proposed that γ-HBCD isomerizes to α-HBCD under 

physiological conditions, rather than uptake being diastereisomer-specific. To test this theory, 

Esslinger et al. (2010) exposed mirror carp (Cyprinus carpio morpha noblis) to only γ-HBCD and 

found no evidence of bioisomerization. In contrast, when Du et al. (2012b) exposed zebrafish 

(Danio rerio) to only γ-HBCD, they found detectable levels of α-HBCD in fish tissue, suggesting 

that bioisomerization occurred. Marvin et al. (2011) hypothesized that differences in accumulation 

could also be due in part to a combination of differences in solubility, bioavailability, and uptake 

and depuration kinetics. 

Zhang et al. (2014c) calculated diastereoisomer-specific BCFs in algae and cyanobacteria ranging 

from 174 to 469. For the cyanobacteria (Spirulina subsalsa), the BCF for α-HBCD (350) was higher 

than the BCFs for β-HBCD (270) and γ-HBCD (174). However, for the tested alga (Scenedesmus 

obliquus), the BCF for β-HBCD (469) was higher than that for the other isomers (390 – 407). 

BCFs for HBCD in fish in the peer-reviewed literature range as high as 18,100, as shown in Table 

4-3 (Drottar and Krueger, 2000; Veith et al., 1979; Zhang et al., 2014b). Drottar and Krueger (2000) 

provided strong evidence that HBCD bioaccumulates in a bioconcentration test that was conducted 

according to guidelines OECD Test Guideline (TG) 305 and Office of Prevention, Pesticides and 

Toxic Substances (OPPTS) 850.1730. In this study, BCFs of 13,085 and 8,974 were reported in 

rainbow trout (O. mykiss) exposed to 0.18 and 1.8 μg HBCD/L, respectively. Concentrations of 

HBCD in tissue reached steady-state at day 14 for fish exposed to 1.8 μg HBCD/L and, during the 

subsequent depuration stage, a 50% reduction of HBCD from edible and non-edible tissue and 

whole fish was reported on days 19 and 20 post-exposure. In fish exposed to 0.18 μg HBCD/L, an 

apparent steady-state was reached on day 21, but on day 35, the tissue concentration of HBCD in 

fish increased noticeably; thus, steady-state was not achieved according to study authors, and BCF 

values (for the exposure concentration of 0.18 μg/L) were calculated based on day 35 tissue 

concentrations. Clearance of 50% HBCD from tissue of 0.18 μg HBCD/L exposed fish occurred 30 

– 35 days post-exposure. 

Veith et al. (1979) further supports a conclusion that HBCD bioaccumulates in a study conducted 

prior to the establishment of standardized testing guidelines for bioconcentration studies. The study 

reported a BCF of 18,100 following exposure of fathead minnow to 6.2 μg HBCD/L; the BCF was 

identified as a steady-state BCF, but the report does not indicate time when steady-state was 

reached. A depuration phase was not included in this study. Zhang et al. (2014b) calculated BCFs 

for each diastereoisomer in mirror carp and found strong evidence that α-HBCD (BCF of 5,570 –

11,500) is much more bioaccumulative than β- and γ-HBCD (BCF of 187 – 642); BCF values that 

were normalized to lipid content were much higher (30,700 – 45,200 for α-HBCD, 1,030 – 1,900 
55

for β-HBCD, and 950 – 1,730 for γ-HBCD) than non-normalized BCFs.

BAFs, which capture accumulation of HBCD from diet as well as water and sediment, were 

calculated for freshwater food webs in industrialized areas of Southern China in two separate field 

studies. He et al. (2013) calculated log BAFs of 4.8 – 7.7 (corresponding to BAFs of 63,000 –

50,000,000) for HBCD isomers in carp, tilapia, and catfish, and found higher BAFs for α-HBCD 

than β- and γ-HBCD. In a pond near an e-waste recycling site, Wu et al. (2011) calculated log BAFs 

of 2.85 – 5.98 for ƩHBCD (corresponding to BAFs of 700 – 950,000) in a freshwater food web. 

Log BAFs for each diastereoisomer in this study were comparable to one another (see Table 4-3). 

La Guardia et al. (2012) calculated log BAFs in bivalves and gastropods collected downstream of a 

textile manufacturing outfall; these ranged from 4.2 to 5.3 for α- and β-HBCD (BAFs of 16,000 –

200,000), and from 3.2 to 4.8 for γ-HBCD (BAFs of 1,600 – 63,000). 

BSAFs calculated in studies of invertebrates and fish are generally lower than reported BCFs and 

BAFs. Haukås et al. (2010b) reported BSAFs ≤0.006 calculated from lipid-normalized 

concentrations of HBCD in ragworms and HBCD concentrations normalized to total organic 

content in sediment, indicating very low bioavailability of HBCD from sediments. Ragworm tissue 

concentrations were all less than the limit of detection. The pattern of diastereoisomers in sediments 

was found to generally resemble the composition of technical HBCD (i.e., predominantly γ-HBCD). 

This study also found that in ragworms exposed to HBCD through a diet of contaminated mussels 

(containing diastereomer contributions of 48% α-HBCD, 7% β-HBCD, and 45% g-HBCD), the 

tissue concentration of α-HBCD was greater than that of β-HBCD or γ-HBCD, suggesting selective 

bioaccumulation of the α-diastereoisomer. 

Log BSAFs calculated in bivalves and gastropods collected downstream of a textile manufacturing 

outfall ranged from 0 to 0.9 (for α- and β-HBCD) and from -1.5 to 0 (for γ-HBCD) (La Guardia et 

al., 2012). These correspond to BSAFs of 1 – 8 for α- and β-HBCD and 0.03 – 1 for γ-HBCD. 

BSAFs in benthivorous barbell (Barbus graellsii) and pelagic bleak (Alburnus alburnus) were 

calculated based on measured concentrations of HBCD reported in Eljarrat et al. (2004, 2005 as 

cited in van Beusekom et al., 2006) and ranged from 0.1 to 1.44 and from 0.14 to 1.23, respectively 

(van Beusekom et al., 2006).

In Haukås et al. (2009), juvenile rainbow trout exposed via a single gavage dose of 0.61 mg 

HBCD/g wet weight (measured) reached a maximum accumulation of HBCD by day 8, with 

considerable amounts of all diastereomers distributed to liver, brain, and muscle. Assimilation 

efficiencies of HBCD for individual diastereoisomers (percent of HBCD isomer in the fish relative 

to the amount in the bolus fed to the fish at the start of the experiment) were >100% for β-HBCD, 

78% for α-HBCD, and 46% for γ-HBCD; values were calculated on day 4. A 71% reduction in 

HBCD was observed by day 21, primarily from the brain and liver. No clear elimination was 

observed from muscles. In zebrafish exposed to HBCD for 42 days, Du et al. (2012b) measured 

diastereoisomer-specific assimilation efficiencies of 40.84 – 54.67% for α-HBCD, 33.59 – 47.71% 

for β-HBCD, and 29.31 – 40.80% for γ-HBCD. Du et al. (2013) reported similar assimilation 

efficiencies in zebrafish (see Table 4-3). Haukås et al. (2010b) reported a 28-day assimilation 

efficiency of 67% in ragworms.

Biomagnification of HBCD was demonstrated by Law et al. (2006), who reported BMFs of 9.2 (αHBCD), 4.3 (β-HBCD), and 7.2 (γ-HBCD). Uptake of HBCD into muscle from the diet of rainbow 
56

trout was exponential for α-HBCD with a doubling time of 8.2 days, exponential for β-HBCD with 

a doubling time of 17.1 days, and linear for γ-HBCD with a rate constant of 0.006 per day. 

Depuration was rapid during the first 14 days and slower for the remainder of the experiment for αHBCD (overall depuration rate was not determined). Depuration rates of 0.44×10-2

and 0.48×10-2

per day were found for β-HBCD and γ-HBCD, respectively. Steady-state was not reached for any of 

the diastereoisomers within the 52-day exposure period. 

Additional studies are available that support the conclusion that HBCD has the potential to 

biomagnify. Studies of zebrafish by Du et al. (2012b, 2013) reported diastereo- and enantiomerspecific biomagnification. When BMFs were calculated for diastereoisomers without accounting for 

specific enantiomers, after 42 days of exposure and a 21-day depuration period, α-HBCD was 

shown to biomagnify to a greater extent than β- and γ-HBCD (maximum BMFs of 29.71, 11.63, and 

7.76, respectively). Enantiomer-specific BMFs calculated in zebrafish by Du et al. (2013) followed 

a similar diastereoisomer pattern, although the BMF values were much lower than those from Du et 

al. (2012b). Additionally, the results of Du et al. (2013) suggest that the (+) enantiomers of β- and γHBCD are selectively magnified compared to their (−) enantiomers. This pattern did not hold true 

for α-HBCD. 

Letcher et al. (2009) found evidence of biomagnification of HBCD from the ringed seal to the polar 

bear in an East Greenland food web, reporting a BMF of 1.7. BMFs for α-HBCD in a harbor seal 

food web varied according to prey fish species, but ranged from 0.54 to 3.0 (Shaw et al., 2012). 

Shaw et al. (2012) calculated higher BMFs from prey fish to the livers of adult male harbor seals 

than to the blubber of those seals. 

BMFs for α-HBCD in gulls and common eiders in a coastal marine food web in Norway provide 

evidence of biomagnification, ranging from 3.1 to 1,285 when calculated on a wet weight basis and 

from 2.8 to 26 when calculated on a lipid-weight basis (Haukås et al., 2010a). In terrestrial food 

webs in China, both Sun et al. (2012) and Yu et al. (2013) found evidence of biomagnification (see 

Table 4-3), with BMFs up to 30 in passerine birds and up to 16 in owls. Yu et al. (2013) found more 

( ̶ ) α-HBCD in predator species than (+) α-HBCD, but other studies do not agree, suggesting that 

enantiomer biomagnification may be species-specific.

Tomy et al. (2008) describes the extent of trophic transfer (transfer and accumulation of HBCD 

between trophic levels) by calculating TMFs of 2.1 and 0.5 for α- and γ-HBCD, respectively, based 

on the Arctic marine food web. Samples of blubber were taken and analyzed from the beluga whale 

(Delphinapterus leucas), narwhal (Monodon monoceros), and walrus (Odobenus rosmarus), while 

whole organisms were analyzed for arctic cod (Boreogadus saida), shrimp (Pandalus borealis and

Hymenodora glacialis), clams (Mya truncate and Serripes groenlandica), deepwater redfish 

(Sebastes mentella), and mixed zooplankton to determine HBCD concentrations in the tissue of 

animals of different trophic levels in order to establish whether HBCD biomagnifies between 

trophic levels. 

Brandsma et al. (2015) studied trophic magnification of HBCD through benthic and pelagic food 

webs in the Western Scheldt estuary, The Netherlands, and found similar results: α-HBCD 

concentrations increased and γ-HBCD concentrations decreased with an increase in trophic level 

(TMFs of 2.2 and 0.3, respectively). In a freshwater food web studied near an e-waste recycling site 

in South China, Wu et al. (2010) calculated enantiomer-specific TMFs for α-HBCD of 2.18 – 2.2, 
57

and found evidence that as HBCD migrates up through the food web, α-HBCD increases and γHBCD decreases, while β-HBCD comprises a very low proportion of ∑HBCD. This pattern, also 

demonstrated by data in Haukås et al. (2010a), becomes more prominent at upper trophic levels. In 

marine and freshwater food webs, Zhang et al. (2013) calculated TMFs greater than 1 for α-HBCD 

and ƩHBCD.

In summary, while HBCD has been shown in numerous studies to bioaccumulate and biomagnify in 

aquatic and terrestrial food chains, diastereoisomer- and enantiomer-specific mechanisms of 

accumulation are still unclear.”

Peer Reviewer 8 Comment: HBCD has significant potential for bioaccumulation through direct 

and indirect (trophic) mechanisms. The extremely high log Kow indicates that accumulation may 

not only occur through direct branchial or dermal routes of exposure in aquatic organisms, but also 

via dietary exposure, particularly in detritivores consuming DOC-bound material, or sediment 

consuming benthic infauna. I agree with the conclusions presented and the data upon which the 

conclusions are based.

EPA Response: Commenter agrees with EPA’s assessment, no action taken or required.

Peer Reviewer 9 Comment: The report cites a number of studies to support the assertion that 

HBCD has the potential to bioaccumulate. While I agree with the claims made in the report, the 

material is not presented in the most user friendly fashion. Most of my concerns revolve around the 

issue of how the material is organized. The authors discuss topics such as BCF, BAF, MF, BSAFs 

and TMF yet the ordering of the information and the interrelatedness of the measures is not 

discussed.

EPA Response: This section was reorganized and rewritten to improve clarity. The section has 

also been expanded to include a discussion of the interrelatedness of the different measures of 

bioaccumulation. In addition, a new literature search has been performed to update the references 

through 2015. Extensively revised text and updated references were included a previous response.

Peer Reviewer 9 Comment: It is not clear why some data are included, yet others are not reported. 

The issues of the whether the studies are conducted in accordance with EPA or OECD guidelines is 

mentioned but the information afforded by and the inherent limitations of non-guideline studies are 

largely ignored. I expected a more focused discussion of the information available for various 

enantiomers.

EPA Response: The literature has been updated and discussion expanded to include additional 

enantiomer-specific data. In addition, the discussion now includes more information on the 

limitations of the various studies, including recent diastereomer-specific bioaccumulation data. 

Extensively revised text and updated references were included in a previous response. 

Peer Reviewer 9 Comments: There was specific mention in the text on page 91 that BCFs of 

13,085 and 8,794 were reported for tests conducted by Drottar and Krueger (2000), presumably 

involving the fathead minnow. The data only lines up in part with the values reported in Table 4.3 

(page 90). Why the discrepancy? 
58

EPA Response: The BCF’s taken from Drottar and Krueger (2000) are based on tests conducted 

on the rainbow trout (Oncorhynchus mykiss): Low exposure group (0.18 μg/L) (BCF – 13,085), 

High exposure group (1.8 μg/L) (BCF: 8,974). Footnote e from Table 4-3 explains that steady state 

was not reached in the low exposure group. Nevertheless the value of 13,085 was added to Table 4-

3 as this is close to the BCF of 18,100 determined by Veith et al. (1979).

Peer Reviewer 9 Comments: A number of other studies have reported various measures that 

describe the bioaccumulation potential for HBCD. Why are these studies not included? While these 

studies may not have been done to an accepted test guideline, they could have been reported as 

being supportive of the overall claim. Perhaps an explicit statement up front on the criteria for 

selection of the data and for exclusions would be appropriate.

Here are some studies that might be considered for inclusion. 

Zhang Y1, Sun H2, Ruan Y1. 2014. Enantiomer-specific accumulation, depuration, metabolization 

and isomerization of hexabromocyclododecane (HBCD) diastereomers in mirror carp from 

water. J Hazard Mater. 264: 8-15. 

Du M, Lin L, Yan C, Wang C, Zhang X. 2013. Enantiomer-specific bioaccumulation and 

depuration of hexabromocyclododecanes in zebrafish (Danio rerio). J Hazard Mater. 248-

249:167-71. 

Du M, Lin L, Yan C, Zhang X. 2012. Diastereoisomer- and enantiomer-specific accumulation, 

depuration, and bioisomerization of hexabromocyclododecanes in zebrafish (Danio rerio). 

Environ. Sci.Technol. 46(20): 11040-6. 

Nyholm JR1, Norman A, Norrgren L, Haglund P, Andersson PL. 2008. Maternal transfer of 

brominated flame retardants in zebrafish (Danio rerio). Chemosphere. 73(2):203-8. 

Zhang X1, Yang F, Luo C, Wen S, Zhang X, Xu Y. 2009. Bioaccumulative characteristics of 

hexabromocyclododecanes in freshwater species from an electronic waste recycling area in 

China. Chemosphere. 76 (11):1572-8.

EPA Response: The literature has been updated and additional peer-reviewed bioaccumulation 

data has been added to cover work up through 2014 (Table 4-3). The 5 studies suggested the 

commenter were examined and included as appropriate. Additional new references cited in 

previous responses were also included.

3. Are there additional key published studies or publicly available scientific reports that are 

missing from the draft document that might be useful for discussion of the ecological hazards? 

Peer Reviewer 7 Comment: Marteinson, S.C., Kimmins, S., Letcher, R.J., Palace, V.P., Bird, 

D.M., Ritchie, I.J., Fernie, K.J., 2011. Diet exposure to technical hexabromocyclododecane 

(HBCD) affects testes and circulating testosterone and thyroxine levels in American kestrels (Falco

sparverius). Environ. Res. 111, 1116-1123.

 0.51 μg HBCD/g kestrel/day

 dissolved in safflower oil (0.32 μg/μl or 800 ppb) and injected into the brains of the dead 

cockerel feed daily

 ad libitum feeding

 authors report that the HBCD concentration was environmentally relevant based on the 

levels recorded in the eggs of wild peregrine falcons (F. peregrinus) (Guerra et al., in 
59

press (Guerra, P., Alaee, M., Jiménez, B., Pacepavicius, G., Marvin, C., MacInnis, G., 

Eljarrat, E., Barceló, D., Champoux, L., Fernie, K.J. 2012. Emerging and historical 

brominated flame retardants in Peregrine Falcon (Falco peregrinus) eggs from Canada 

and Spain. Environ. Int., 40, 179-186).

 Testes size altered in exposed birds relative to controls

 Plasma T4 reduced throughout study in exposed birds

Marteinson, S.C., Bird, D.M., Letcher, R.J., Sullivan, K.M., Ritchie, I.J., Fernie, K.J. 2012. Dietary 

exposure to technical hexabromocyclododecane (HBCD) alters courtship, incubation and parental 

behaviors in American kestrels (Falco sparverius). Chemosphere 89, 1077-1083. 

 0.51 μg HBCD/g kestrel/day

 dissolved in safflower oil (0.32 μg/μl or 800 ppb) and injected into the brains of the dead 

cockerel feed daily

 4 wk exposure prior to mate pairing

 reduced courtship

 reduced parental care in males

 nest temperatures altered

Zhang, H., Pan, L., Tao, Y., 2014. Antioxidant responses in clam Venerupis philippinarum exposed 

to environmental pollutant hexabromocyclododecane. Environ. Sci. Pollut. Res. Int. 21, 8206-8215.

 0.086, 0.86, and 8.6 μg/L nominal concentrations of HBCD

 dissolved in DMSO, final concentration = 0.001%

 actual exposure concentrations not measured

 1-, 3-, 6-, 10-, and 15-day exposure to seawater containing HBCD

 Activities of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), 

superoxide dismutase (SOD), and glutathione (GSH), as well as DNA damage and lipid 

peroxidation (LPO) were found in in gills and digestive glands of exposed clams.

Ronisz, D., Finne, E.F., Karlsson, H., Förlin, L., 2004. Effects of the brominated flame retardants 

hexabromocyclododecane (HBCDD), and tetrabromobisphenol A (TBBPA), on hepatic enzymes 

and other biomarkers in juvenile rainbow trout and feral eelpout. Aquat Toxicol. 69, 229-245.

 5- and 28 days experiments using juvenile trout

 50 and <500 mg/kg injected i.p. on days 1 and 14

 peanut oil used as solvent vehicle

 measured 0.14–0.7 ug/g for the 50 mg dose and 7–15 ug/g for the 500 mg dose in bile 

after the exposures.

 in vitro studies using 0.5 mg HBCD dissolved in 50 ul DMSO.

 40% increase in liver somatic index (LSI) could be observed in exposed trout after 28 d

 HBCD lowered EROD activity at the < 500 mg/kg dose

Zhang, Y., Sun, H., Ruan, Y., 2014. Enantiomer-specific accumulation, depuration, metabolization 

and isomerization of hexabromocyclododecane (HBCD) diastereomers in mirror carp from water. J. 

Hazard. Mater. 264, 8-15.

 HBCD diastereomers separated by HPLC, speed-vac'd, reconstituted in MeOH. Whether 

this was used as a vehicle for exposure studies is not clear. 

 30 d uptake, 30 d depuration, 1 ng/L exposure concentration. Vehicle??

 rank order for enantiomer accumulation was viscera > gill > skin > muscle

 BCFs of α-HBCD in different tissues of the carp were in the range of (3.07–4.52) × 104, 

much higher than those of β-HBCD (1.03–1.90 × 103) and γ-HBCD (0.95–1.73 × 103).
60

Zhang, Y., Sun, H., Zhu, H., Ruan, Y., Liu, F., Liu, X., 2014. Accumulation of 

hexabromocyclododecane diastereomers and enantiomers in two microalgae, Spirulina subsalsa and 

Scenedesmus obliquus. Ecotoxicol. Environ. Saf. 104, 136-142. 

 exposure concentration was 2 ng/mL

 In S. subsalsa, the BCFs for α-, β-, and γ-HBCD were 350, 270, and174, respectively, 

whereas in S. obliquus, the corresponding BCFs for α-, β-, and γ-HBCD were 407, 469, 

and 390, respectively.

Malarvannan, G., Belpaire, C., Geeraerts, C., Eulaers, I., Neels, H., Covaci, A., 2014. Assessment 

of persistent brominated and chlorinated organic contaminants in the European eel (Anguilla 

anguilla) in Flanders, Belgium: Levels, profiles and health risk. Sci. Total Environ. 14, 482-483, 

222-233.

 Fish collected from 60 sites in Belgium.

 For HBCDs, α-HBCD (74%) was predominant followed by γ-(22%) and β-HBCD (4%) 

isomers in almost all eel samples.

EPA Response: EPA reviewed, added and expanded the discussion to include all studies suggested 

by the commenter. The bioaccumulation section has been extensively revised and expanded. See 

previous response to comments on question 2 above.

Peer Reviewer 8 Comment: Several publications that may be helpful include:

1. Fernie et al., 2011. (Environmental Toxicology and Chemistry 30:2570-2575) 

In this study, American kestrels were exposed daily by food to HBCD-TM, i.e., 0.51 g HBCD/g 

kestrel/d; exposed kestrels laid eggs that had -HBCD concentrations (163.5+75.1 ng/g wet wt) 

tenfold greater than - and -HBCD isomers. In comparison with controls, the kestrels exposed 

to HBCD began to lay their eggs 6d earlier and laid larger clutches of smaller eggs. The size of 

the eggs was inversely correlated with the in ovo a-HBCD concentrations.

2. Marteinson et al., 2011; Environmental Research 111:1116-1123 and Marteinson et al., 2012 

Chemosphere (2012), 89(9), 1077-1083. 

Demonstrated endocrine disruption and reproductive behavior alterations in kestrels. 

3. Hong et al., 2014; (Aquatic Toxicology 152: 173-185) 

Marine Medaka (fish) exposed to 0, 5, 20 and 50 ug/L for 17 days post fertilization 

demonstrated significant larval deformations in 50 ug/L with approx. 9000 ng/g wwt body 

burden in the embryo.

4. Foekema et al. 2014 (Ecotoxicology Environmental Safety 101:14-22) 

No effects in zebrafish larvae (6 days post fertilization) with 12,500 ug/g lipid wt. body burdens.

5. Jin et al. 2012 In: Global Contamination Trends of Persistent Organic Chemicals (Edited 

by Loganathan, Bommanna G.; Lam, Paul K. S) pp 497-513 

Updates Marine Mammal concentrations.

6. Wu et al., 2013 (Ecotoxicology 22:1200-1207) 

Zebrafish embryos were exposed to HBCD at concns. of 0, 2, 20 and 200 nM. Increase in heart 

rate and cardiac arrhythmia after exposure for 72 h, though the survival rate and the whole 

malformation rates were not significantly affected.

7. Du et al., 2012 (Aquatic Toxicology 112-113:1-10) 

Four-hour post-fertilization (hpf) zebrafish embryos were exposed to different concns. of HBCD 

diastereoisomers (0, 0.01, 0.1 and 1.0 mg/l) until 120 hpf. significant increase in mortality and 

malformation rate was noted at 0.1 mg/l γ-HBCD exposure groups (P < 0.05). At 0.1 mg/l, αHBCD resulted in depressed heart rate of larvae (96 hpf) and delayed hatching, whereas β- and 
61

γ-HBCD both caused significant hatching delay and growth inhibition (P < 0.05). 

Developmental toxicity of HBCD diastereoisomers in zebrafish is γ-HBCD > β-HBCD > αHBCD.

EPA Response: EPA reviewed, added and expanded the discussion to include all studies suggested 

by the commenter. The bioaccumulation section has been extensively revised and expanded. See 

previous response to comments on question 2 above.

Peer Reviewer 9 Comment: As mentioned above it is not clear what criteria are being used for 

determining which studies are reported. A quick search of published studies identified a significant 

number of studies that were not included in the report. While the report mentions that many 

responses are seen in fish and invertebrates when studies are conducted based using protocols that 

deviate from guideline studies (starting bottom of page 67), the list of responses is not exhaustive. It 

is not clear why some studies are included and others are not. Here are some studies that might have 

added to the discussion. 

Anselmo HM, Koerting L, Devito S, van den Berg JH, Dubbeldam M, Kwadijk C, Murk AJ. 2011. 

Early life developmental effects of marine persistent organic pollutants on the sea urchin 

Psammechinus miliaris Ecotoxicol Environ Saf. 74(8):2182-92. Erratum in: Ecotoxicol Environ 

Saf. 80:401. 

Crump D, Egloff C, Chiu S, Letcher RJ, Chu S, Kennedy SW. 2010. Pipping success, isomerspecific accumulation, and hepatic mRNA expression in chicken embryos exposed to HBCD.

Toxicol Sci.;115 (2):492-500.

Deng J1, Yu L, Liu C, Yu K, Shi X, Yeung LW, Lam PK, Wu RS, Zhou B. 2009. 

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos. 

Aquat Toxicol. 2009 93(1):29-36.

Fernie KJ, Marteinson SC, Bird DM, Ritchie IJ, Letcher RJ. 2011. Reproductive changes in 

American kestrels (Falco sparverius) in relation to exposure to technical 

hexabromocyclododecane flame retardant.Environ Toxicol Chem. 30(11): 2570-5.

Hu J, Liang Y, Chen M, Wang X. 2009. Assessing the toxicity of TBBPA and HBCD by zebrafish 

embryo toxicity assay and biomarker analysis. Environ Toxicol. 24(4):334-42

Kling P, Förlin L. 2009. Proteomic studies in zebrafish liver cells exposed to the brominated flame 

retardants HBCD and TBBPA. Ecotoxicol Environ Saf. 72(7): 1985-93. 

Luo XJ, Ruan W, Zeng YH, Liu HY, Chen SJ, Wu JP, Mai BX. 2013. Trophic dynamics of 

hexabromocyclododecane diastereomers and enantiomers in fish in a laboratory feeding study.

Environ Toxicol Chem. 32(11):2565-70.

Marteinson SC, Bird DM, Letcher RJ, Sullivan KM, Ritchie IJ, Fernie KJ. 2012. Dietary exposure 

to technical hexabromocyclododecane (HBCD) alters courtship, incubation and parental 

behaviors in American kestrels (Falco sparverius). Chemosphere. 89 (9):1077-83.

Marteinson SC, Kimmins S, Letcher RJ, Palace VP, Bird DM, Ritchie IJ, Fernie KJ.2011. Diet 

exposure to technical hexabromocyclododecane (HBCD) affects testes and circulating 

testosterone and thyroxine levels in American kestrels (Falco sparverius).Environ Res. 

111(8):1116-23.

Schriks M, Zvinavashe E, Furlow JD, Murk AJ.2006. Disruption of thyroid hormone-mediated 

Xenopus laevis tadpole tail tip regression by hexabromocyclododecane (HBCD) and 

2,2',3,3',4,4',5,5',6-nona brominated diphenyl ether (BDE206). Chemosphere 65(10):1904-8. 
62

EPA Response: EPA reviewed, added and expanded the discussion to include all relevant studies

cited by the commenter.

4. Please provide any additional comments or suggestions you may have that may improve 

this section of the document.

Peer Reviewer 7 Comment: The authors consistently refer to 'low' versus 'small' concentrations. 

Also the authors should delete 'very' (p 67, line 12 for example) when referring to measured 

concentrations or endpoints as 'very' has no units of measurement and suggests a subjective 

evaluation of the measurement.

EPA Response: EPA added units and supporting quantitative values.

Peer Reviewer 7 Comment: p 67, line 13, change 'conduct of the tests' to 'testing'.

EPA Response: EPA made the change.

Peer Reviewer 7 Comment: p 68, the last paragraph of the summary is redundant.

EPA Response: Text was edited to remove redundancy.

Peer Reviewer 7 Comment: p 69, what is the point of including half a paragraph on the OECD 

guidelines when the authors never explain the relevance to the current assessment. Were these 

guidelines followed and /or incorporated into the assessment?

EPA Response: EPA edited the text to clarify relevance of including discussion of OECD 

guidelines in terms of results of ecotoxicity testing. EPA also added a discussion concerning the 

relevance of solvent concentrations at or above the reported water solubility limit and relevance to 

study reliability.

Peer Reviewer 7 Comment: p 70, line 10, include a citation and reference to support the 

'Environmental monitoring data...' that are being discussed.

EPA Response: EPA added a citation string referring to the monitoring data (Allchin and Morris, 

2003; De Boer et al., 2004; Zegers et al., 2005; Muir et al., 2006; Tomy et al., 2008).

Peer Reviewer 7 Comment: Table 4.1, p 77. What constitutes an 'invalid' study? Suggests that 

some type of criteria were used to weigh the strengths of the data presented in each paper, but never 

indicate what this is.

EPA Response: EPA edited and expanded the ‘Comments’ section of Table 4.1 to clarify why the 

data were considered invalid.

Peer Reviewer 7 Comment: Table 4.1, update with more recent references as appropriate.

EPA Response: Additional data and studies were added and Table 4.1 was extensively updated 

with more recent references.
63

Peer Reviewer 7 Comment: p 82, missing references for Table 4.1. Maybe a word processing 

issue where the table is folded over into the next page but the reference (s) should be included. p. 

83, Table 4.1, missing reference.

EPA Response: The reference (Porch et al., 2002) was included in the table, but the record was 

split across several pages, text was edited to fix and clarify.

Peer Reviewer 7 Comment: p 86, Hamers et al. (2006), what species?

EPA Response: Hamers et al., 2006 is an in vitro study that included several bioassays that used 

various cell types or protein receptors. The cells and protein receptors used in this study were 

derived from mammals (rats and humans), therefore the text was moved to the Human Health 

chapter and clarified.

Peer Reviewer 7 Comment: p 88, line 2, what is a 'corticosterone response'? Cort is a hormone 

and doesn't respond to anything. Do the authors mean secretion of cort? Cort action at a receptor? 

Please clarify.

EPA Response: The “corticosterone response” mentioned in Table 4-1 (page 81) and on page 88 

refers to the study conducted by Kobiliris et al. (Kobiliris, D. Unpublished. Influence of embryonic 

exposure to hexabromocyclododecane (HBCD) on the corticosterone response and “fight or flight” 

behaviors of captive American kestrels. Accessed at http://www.researchgate.net).

Text has been expanded to include clarification of the ‘corticosterone response’ including addition 

of a more recent citation of published graduate work (Kobiliris, 2010).

Peer Reviewer 7 Comment: p 92, line 4, change 'establish' to 'established'.

EPA Response: EPA made the correction.

Peer Reviewer 7 Comment: references, please check carefully to italicize genus and species 

names, for example Kuiper et al., p 95.

EPA Response: EPA reviewed and corrected the references.

Peer Reviewer 8 Comment: Given the accumulation kinetics of HBCD, attempts should be made 

to use critical body-burden concentrations for toxicity thresholds for oviparous species and back 

calculate to aqueous or sediment concentrations for regulatory purposes (Meador et al., 2010; Beyer 

and Meador 2012). This method has been very helpful for bird (Selenium; Mercury) and fish (TEQs 

for dioxin-like compounds; PCBs; Selenium) development and/or reproduction estimates based on 

egg or body burden concentrations.

Beyer, WN; Meador, JP (2011) Environmental Contaminants in Biota (2nd Edition) CRC Press 

Boca Raton, FL 1-8pp.
64

Meador et al. (2010). Bioaccumulation of polychlorinated biphenyls in juvenile chinook salmon 

(Oncorhynchus tshawytscha) outmigrating through a contaminated urban estuary: dynamics and 

application. Ecotoxicology 19:141-152.

EPA Response: The above 2 studies were reviewed for inclusion in the hazard assessment. 

However, both are primarily focused on discussion of the regulation of HBCD which is outside the 

scope of the hazard assessment.

Peer Reviewer 9 Comment: The text would have benefitted from a brief synopsis section at the 

end of the major sections (e.g., 4.2 and 4.3). It seemed that the sections ended abruptly and would 

have benefitted from some concluding remarks or a statement of data gaps.

EPA Response: Section 4.4 Conclusions was added to summarize the overall ecotoxicity and 

bioaccumulation potential for HBCD as follows:

“There is evidence for high concern for both acute and chronic toxicity to aquatic and terrestrial 

organisms exposed to HBCD. HBCD can cause effects on survival, growth, reproduction, 

development, and behavior. The potential of HBCD to bioaccumulate in aquatic and terrestrial 

organisms and biomagnify in food webs is high.”