# Response to the External Peer Review of

“Technical Review of Hexabromocyclododecane”

January 11 , 201 6

United States Environmental Protection Agency Office of Environmental Information Office of Information Access and Analysis2 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 H BCD 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 result ing in the formation of toxic chemicals?.....................................................................................

............ ............. 9 4. Please provide any additional comments or suggestions you may have that may improve this se ction of the technical review. ...............

......... .......1 3

Response to External Peer Review Section II: Human Health Effects

........................ . ..1 7

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

........... .... 1 7 II. Response t o Human Health Hazard Assessment Charge

.. ... .............. ............ .......... . ... 1 7 A. General Impressions.........................................................

........... ......... 1 7 B. Peer Reviewer Comments by Charge Question............

. ............ ......... ...... .. .... 1 7 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?

........................... ................. ... ......... ....................... ....... 1 7 2 a . Please comment on the non

- cancer endpoints. Has the scientific evi dence for

non

- cancer endpoints been accurately and clearly represented and evaluated? Do
you agre e or disagree with the conclusions?

...................... ............................... ..... 2 4 2b

. Please comment on the adequacy of the conclusions in the HBCD hazard assessment with rega rd 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.

.... .... 3 2 3

. Do you agree or disagree with the selection of the LOAELs/NOAELs (or BMDLs/BMDs) for all endpoints?

. ................................................................ .... 3 5 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 discussio n 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.

............................................. .....4 23

Response to External Peer Review Section III: Ecological Hazard Assessmen t

......... .... ...4 5

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

........ ...................... 4 5 II. Response to Ecol ogical 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?

................................................................ .......................................... . 5 3 3. Are there additional key published studies or publ icly 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 commen ts or suggestions you may have that may improve this section of the document.

..................... ............................ . 6 24 External Peer Review of EPA’s Draft Technical Assessment of

Hexabromocyclododecane (HBCD )

BACKGROUND

Hexabromocyclododecane (HBCD) is not currently listed o n the Toxic s 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 atom s 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 techni cal 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 a s 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 ungrade d 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 EPA5

cited 28 does not mean that EPA missed relevant information. Some of the re view 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 19 0

o C the diastereo mers 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 differ ent compositions of diastereo mers; 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 hav e 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 F lame 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. E dited 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 Value a Reference
Molecular formula C 12 H 18 Br 6

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 s olubility 8.6 μg/L at 25 °C ( 14 C

- 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 K ow 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

K oc 7.6×10 4 (estimated) U.S. EPA, 2011b (EPISuite TM v4.10) Henry’s Law
constant 4.6×10

- 5 atm

- m 3 /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/cm 3

2.38 g/cm 3 (technical product) Great Lakes Chemical Corporation, 1994 and Albemarle Corporation, 1994 as cited in ECB, 2008 Flash point Not applicable

a Values 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 co mmercial 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 stereo isomer 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, Un ited 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 diff

erence 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 interco nversion 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 wit h 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 fo od 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 mec hanisms 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 rela ted8

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 environmen tal 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 was tewater 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 172 o C

- 209 o C (Table 2

- 1). Yet the EPI Suite calculation uses the temperature 190 o C with no justification presented for the selection. In fact the same table reports decomposition at temperatures in excess of 190 o C. 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 dis cussion clarifying the issue of thermal degradation, as discussed in the primary source Peled et al. 1995. At 190 o C, the diastereoisomers began to thermally re

- arrange, and decomposition was reported as the temperature in which 5% of the original materia l 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 distri bution 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 en hanced 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 dir ectly 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 whethe r 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 adde d 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 H BCDs 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 isome r 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 diaster eomers.

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 th e 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 conc epts 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

- 5 atm

- m 3 /mol. ” This statement is factually incorrect, in that the10

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

wa ters 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 es timate 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

- m 3 /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 fr om 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
pa rticulates. 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 nuc leophile 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 degrad ation 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 o f 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

- d issolved 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 fro m 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: T he 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 tetrabro mocyclododecadienes (TBCDs)) in a variety of environmental matrices, including lacustrine11

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 fro m 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 show ed 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), Esslin ger 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 Transfor mation 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 i sobutoxypentabromocyclododecanes (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
cy clododecatriene (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 ana lysis (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 wit h 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 me thylmercapturate 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 environ ment 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., 20 06; 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 c ase 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 phy sical 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 inter conversion 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 ster eoisomer 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 d ifference 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 inte rconversion 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 po int, 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 pro perty data should be based on original literature only.

EPA Response: Where possible, physica l 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 hexabromocyclododecane s (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, Wildlif e 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, propertie s 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 descri bes 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 (not

exhaustive) 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 (TBBP

A) 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 I nternational, 35, 573

– 579 (2009b). (7) F. Barontini, V. Cozzani, L. Petarca, “Thermal stability and decomposition products of hexabromo cyclododecane”, 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. S ci. 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 D egradation 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, 19 4

- 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, Zind el D, Geueke B, Kohler HP, Lienemann P. Biotransformation of Hexabromocyclododecanes (HBCD) with LinB

– an HCH

converting bacterial enzyme. Environ. Sci. Technol., 46, 6566

- 6574 (2012). (16) Heeb NV, Zindel D, Schweizer WB, Lienemann P. 2,5,6,9,10

- Pentabromocyc lodecanols (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 re ferences and added those that w ere 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 compoun ds 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 Respons e: EPA added more discussion about the isomers and included the suggest ed 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 rang es 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, were16

calculated using a semi

- empirical method with molecular modeling using the global minimum energy conformer, 1H nuclear magnetic resonanc e (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 s ub

- 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 compa rtments.

“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 thes e stereoisomers. Although δ and ε

- HBCD are rarely detected in environmental compartments, one monitoring study reported detection of δ

HBCD in 13 fish samples from English lakes, but not the associated water or sediment samples (Harrad et al., 2009b). Co nsistent 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: Th e 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 revie w, 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 s cientific 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 t rends, 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 c omment 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 commen ts or suggestions you may have that may improve this section of the document.

II

. Response t o 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 Question s

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 conc ern for moderately high to high toxicity of HBCD.” Nonetheless, the18

major effect of HBCD, developmental neurotoxicity, is missing in the conclusion. The overall conclusion should also include the findings on acute toxicity, carcinogenicity, genotoxicity, t he 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 bod y, 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 is omers can impact on toxicity.

EPA Response: Th e 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 example s of thes e d escriptions 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 da ys. 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 thro ugh 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 stateme nt 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, predomin ately 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 ” f ew 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 t ests 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 of20

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 d ata 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 n either 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 wei ght 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 ma jor 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 di stinct 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 lung s 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 t he 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 an d 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 disti nct 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 art icle 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 metabol ic 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 speci es.
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 simila r 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 re port 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 EP A , n o response required

.

Peer Reviewer 6 Comment: This summary provides an overview of the human health hazard22

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 unclea r 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 measu res. 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 n g/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 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). 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 detec ted (<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 comp rised 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 HB CD 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 r eferences 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

accumu lates 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 refe rence?

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 half

life? 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 non

occupationally exposed adult hum ans 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 i n 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[ 14 C]

- HBCD was eliminated in the urine (30%) and feces (50%) four days following a single gavage dose of 3 mg/kg.”24

2a. Please comme nt 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 prov ides 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 assessmen t. 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 concentra tion of 0.05, 0.5, or 5%. When i nduction of sensitization occurred, challenge doses of 0. 005, 0.05 , 0.5, or 5% (dissolve d 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 th e 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). T hese authors detected HBCD in the maternal serum (n=69) and cord serum (=12) of pregnant women and their infants in the 35 th 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 Res ponse : 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., 20 06) 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 i n 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 t o 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 detect ed 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 wer e 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, w hereas 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 γ

HBCD to α

- HBCD in the liver decreased with increasing exposure concentration (liver lipid

co ncentrations 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?

EP A 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

- lives26

of HBCD isomers in human s: (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 pentabromocyclodo decenes (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

ex posure 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, dihydrox y

pentabromocylcododecene, 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 simplif y 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 r ats 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 a27

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 non

cancer 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, n o 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, n o 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 b e “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” bu t 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 addre ss 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 belo w:

“ 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 group s, 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 Tabl e 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 mal es 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 re lative 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 i n 100 Table 3

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

Species/strain/test Exposure Result Notes Reference
Rat/Charles River/LD 50 4

- hr exposure to

Firemaster 100 dust: 202.14 mg/L No mortality;

s light 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/LC 50 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 gro ss necropsy of rat that died. IRDC, 1978b

Rat/NR/LC 50 test 1 hr. whole

- body

inhalation exposure to GLS

- S6

- 41A LC 50 >200 mg/L No significant effects observed. Gulf South Research Institute, 198829

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 concentrat ions 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 stud y 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 EP A report and I agree with the conclusions regarding the BMDL and the CED.

Cheng e lis (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 Ve n (2006). The latter investigator used more advanced technology and sophisticated data analysis, and the results should be given more weight than those of Cheng e lis (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, n o response required

.

Peer Reviewer 5 Comment: The Cheng e lis et al. (1997) document (EPA HPV Chemical Ass essment) 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 Kirs c h, 1969, 1970). No mention was made of these studies in the EPA Technical Assessment document.

EPA Response : T he se paper s 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 observ ed 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 deve lopment 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 hyperactivi ty 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 n o 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 sign s, 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 r ats, 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 r egards 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 i s (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 kep t together and all the males ones kept together

EPA Response : EPA determined that grouping the findings by endpoint

, rather than by sex provides a clear er 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.

Pee r 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 shorte r term studies or mode

- of

action 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 neuroto xicity 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 R esponse : 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 mechani sms 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: Additi onal 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
cata lepsy...”

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 .”

P eer 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 interva ls. 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, n o re sponse 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 validi ty 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 no t examine toxicity e ndpoints, 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 developmen tal 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 u nit 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 Eu ropean 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% th roughout 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 wer e 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 var iations 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 75 0 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 75033

mg/kg

- day and the maternal LOAEL was 750 mg/kg

- day based on 13% liver weight increase in the high dose g roup."

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 pri mordial 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 particl e 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 shou ld 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 statisti cally 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 decrea ses 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 signif icance 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 st atus, 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 t he 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/da y) 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 id entified. An importan t 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 horm one 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 exper imental unit of analysis. The LOAELS have been appropriately identified in the publication and in the EPA report.

EPA Res ponse : Commenter agrees with EPA, n o 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 decreased34

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 a ny 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/da y 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 a n 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, t he 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 aft er 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 predom inant cochlear effect, possibly influenced by reduced levels of retinoids. Effects were dose

dependent 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 magn itude 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 va lidation 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 n ot seem to be compelling concerns as both types of exposures occur in human populations.

E PA 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

re sult 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.

E PA Response : Commenter agrees with EPA, n o 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 S aegusa 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, based35

on the developmental brain effect, 100ppm was determined to be the no

- observed

- adver se

- 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 le vels 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 “tw enty

- 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 calcu late 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, sh ould 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 fe tal 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 c ontrols 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 a nimal 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 conce ntrations 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 publicatio n. 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 assessmen t 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 as sessment. 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 knowled ge 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 range s to calculate the time

- weighted average dose. Additionally

, this hazard assessment is consistent with EPA ’s use of these time

- weighted average dos es 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, includ ing 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 : Th e change s 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)
Males c Females c
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
T 3 (ng/ml) 1.09 1.13 1.06 0.93 ** ND ND ND ND
T 4 (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
T 3 (ng/ml) 0.96 0.93 0.88 * 0.89 ** ND ND ND ND
T 4 (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 ND38

Abbreviations: BW, body weight; PND, postnatal day; ppm, parts per million; PNW, postnatal week; ND, no data ; NA, not applicable

a Grade of change: (±) minimal; (+) slight; (++) moderate; (+++) severe
b Number of animals with each grade

c n=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 sh owed 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 “measur ed” 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 n ot 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 decr ease 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 significa nt effects on either39

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 t hat 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 highe r 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 5 Comment: I agree with the selection of the LOAELs/NOAELs, BM DLs 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 s hould be given preference in describing human health hazards.

EPA Response : Commenter agrees with EPA, n o 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 re tardants 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 comp lete 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: Relation ship 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. Environmenta l 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, Sza bo 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. He xabromocyclododecane 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., 201 2. 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 adu lt 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 do se, 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 hexabromocycl ododecane 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 Tetrabromobispheno l 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; Hin kson 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 2 006), 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 r elevant 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 single41

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 (Sza bo 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 co mposition 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 product s 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 hu man 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 m etabolites 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 th at 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 f

lame 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. Envir on 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, Schoeter s 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: 1 993

- 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 incorporat ed

.

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, sig nificant 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 expos ure 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 misc lassification 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 t he 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 sent ence.
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 a ddress 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 perio d. 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, w hile human exposure to environmental and dietary contamination, contains elevated fraction of alpha

- HBCD (Schecter et al., 2012) . Alpha

HBCD 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: Her e 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 HBC D 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 t hese studies were not r elevant to add to the human health hazard assessment becau se they describe environmental f ate 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)

E PA Respons e : 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 stu dy, 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 depen dent 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 devel oping 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 incr eased 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 l ower 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 (BMD L). 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 con tradictory 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 li terature. 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

. Charg e 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 bioaccum ulation. 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 dra ft 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 Questio n

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 rega rding 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 thyr oid 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

c onserved, 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 inf ormation 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., develop mental 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 abnormali ties, decreased hatching success, and altered46

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 ( triiodot hyronine, 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 Asses sment) . 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 Review er 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 se ction? 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 fo llowed 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). I f 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 impressi on 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 cas e 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 discuss ion.

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 Co

operation and Development [OECD]), but also non

- standard toxicity tests that fo llowed 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

- observed

effect 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 diastere omer 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 ecotoxi cological 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 solvent s 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 (200 0) 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 conce ntrations 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 dependin g 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 es tablish a more definitive limit of water solubility. ”

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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 redun dant 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 high ly 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, n o 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 biomagni fication 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 aq uatic 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 cle arly 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. H owever more recent 2014 studies (below) should be included and the literature and summary updated accordingly.

EPA Response: The s uggested 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 ass imilation 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 acco unt 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 environ ment.

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 organism54

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 th an 1 indicates potential for biomagnification through the food chain, such that a predator species wo uld 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 fo od webs to a greater extent than the β

- and γ

- diastereoisomers. Uncertainty remains as to the balance of diastereoisomer accumulation in various species a nd the extent to which bioisomerization and biotransformation rates for each isomer affect bioaccumul ation potential. Some authors (e.g., Law et al., 2006) have proposed that γ

- HBCD isomerizes to α

- HBCD under physiological conditions, rather than uptake be ing diastereisomer

- specific. To test this theory, Esslinger et al. (2010) exposed mirror carp ( Cyprin us 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 o ccurred. 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

- spec ific 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 K rueger (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, BC Fs 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

su bsequent 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 f ish 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
repo rted 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 t han β

- and γ

- HBCD (BCF of 187

– 642); BCF values that were normalized to lipid content were much high er (30,700

– 45,200 for α

- HBCD, 1,030

– 1,90055

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 (correspon ding 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 freshwate r 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 m anufacturing 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 BAF s. 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 musse ls (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. BS AFs 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 accumulati on 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 th e fish at the start of the experiment) were >10 0% 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 mu scles. 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 assimil ation 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 (α

HBCD), 4.3 (β

- HBCD), and 7.2 ( γ

- HBCD). Uptake of HBCD into muscle from the diet of rainbow56

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. Depuratio n was rapid during the first 14 days and slower for the remainder of the experiment for α

HBCD (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 wa s 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

- a nd enantiomer

specific 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, r espectively). 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). Additio nally, the results of Du et al. (2013) suggest that the (+) enantiomers of β

- and γ

HBCD 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 H BCD 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) calculate d higher BMFs from prey fish to the livers of a dult 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 wh en 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 B MFs 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) des cribes 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 be luga whale ( D elphinapterus 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 gr oenlandica )

, 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 freshw ater 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 γ

HBCD decreases, whi le 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 e t 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

accu mulation are still unclear. ”

Peer Revi e wer 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 a lso 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 , n o 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 presente d 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 upd ate 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

en antiomers.

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 st udies, including recent diast ere omer

- 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 test s 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 ba sed 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 potenti al 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

se lection of the data and for exclusions would be appropriate.

Here are some studies that might be considered for inclusion.

Zhang Y 1, Sun H 2, Ruan Y 1. 2014. Enantiomer

- spe cific 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 a ccumulation, depuration, and bioisomerization of hexabromocyclododecanes in zebrafish (Danio rerio). Environ. Sci.Technol. 46(20): 11040

- 6. Nyholm JR 1, 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 X 1, Yang F
, Luo C

, Wen S

, Zhang X

, Xu Y . 2009. Bioaccumulat ive 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 res ponses 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 environ mentally relevant based on the levels recorded in the eggs of wild peregrine falcons (F. peregrinus) (Guerra et al., in59

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 e xposed 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 sparveri us ). 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. Ro n isz, D., Finne, E.F., Karlsson, H., Förlin, L., 2004. Effects of the brominated f lame 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 liv er 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 hexabromocyclodode cane (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) × 10 4

, much higher than those of β

- HBCD (1.03

– 1.90 × 10 3 ) a nd γ

- HBCD (0.95

– 1.73 × 10 3 ).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., B elpaire, 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 Enviro n. 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 r eviewed, 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 concentr ations. 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; (Aquati c 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 (Ecotoxic ology 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 exposur e 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, α

HBCD resulted in depressed heart rate of larvae (96 hpf) and delayed hatching, whereas β

- and61

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 > α

HBCD

. EPA Response: EPA r eviewed, 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 inve rtebrates 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 th at might have added to the discussion.

Anse lmo 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 E nviron 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, isomer

specific accumulation, and hepatic m RNA expression in chicken embryos exposed to HBCD. Toxicol Sci.;115 (2):492

- 500. Deng J 1, Yu L
, Liu C

, Yu K
, Shi X

, Yeung LW
, Lam PK
, Wu RS

, Zhou B . 2009. Hexabromocyclododecane

- induced developmental toxicity and apoptosis in zebrafi sh 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. P roteomic 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 ). Chemo sphere. 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
testos terone 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 la evis 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 r eviewed, 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 a dded units and supporting qu antitative 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 e dited 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 t he assessment?

EPA Response: EPA e dited the text to clarify relevance of including discussion of OECD guidelines in terms of results of ecotoxicity testing. EPA also a dded a discussion concerning the relevance of solvent concentrations at or above the r eported 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 a dded 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 e dited 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: Additi onal 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 rec ord was split across several pages, text was e dited 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 typ es 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 'corticoste rone 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 r eviewed 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 concent rations 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 aqua tic and terrestrial organisms and biomagnify in food webs is high.”