&EPA
                                                             EPA/630/R-13/235
                                                            Preliminary Materials
                                                               www. ep a. go v/ir is
 Preliminary Materials for the Integrated Risk Information System (IRIS)
        Toxicological Review of Hexabromocyclododecane (HBCD)

                             [CASRN 3194-55-6]
                                   DRAFT
                                  March 2014
                                   NOTICE

This document is comprised of preliminary materials. This information is distributed solely for
the purpose of pre-dissemination review under applicable information quality guidelines. It has
not been formally disseminated by EPA. It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
                    National Center for Environmental Assessment
                         Office of Research and Development
                        U.S. Environmental Protection Agency
                                Washington, DC

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                            Preliminary Materials for the IRIS Toxicological Review ofHBCD
                                      DISCLAIMER
       This document is comprised of preliminary materials for review purposes only. This
information is distributed solely for the purpose of pre-dissemination review under applicable
information quality guidelines. It has not been formally disseminated by EPA. It does not represent
and should not be construed to represent any Agency determination or policy. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
          This document is a draft for review purposes only and does not constitute Agency policy.
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                             Preliminary Materials for the IRIS Toxicological Review ofHBCD
CONTENTS
PREFACE	v
1.   PLANNING AND SCOPING SUMMARY	1-1
    l.l.HBCD Chemistry and Uses	1-1
    1.2.HBCD in the Environment	1-2
    1.3. Rationale for the Development of the Toxicological Review	1-3
    1.4. General Scope of the Toxicological Review	1-4
2.   DRAFT LITERATURE SEARCH AND SCREENING STRATEGY	2-1
3.   SELECTION  OF STUDIES FOR HAZARD IDENTIFICATION	3-1
    3.1.General Approach	3-1
    3.2. Selection  of Primary Studies for Evidence Tables for HBCD	3-2
       3.2.1. Epidemiologic Studies	3-2
       3.2.2. Experimental Animal Studies	3-3
    3.3. Preliminary Evidence Tables and Exposure-Response Arrays	3-3
    3.4. Study Characteristics That Will Be Considered in the Evaluation and Synthesis of the
       Primary Studies for HBCD	3-4
       3.4.1. Epidemiologic Studies	3-4
       3.4.2. Experimental Animal Studies	3-8
4.   REFERENCE LIST	4-1
APPENDIX A. PRELIMINARY EVIDENCE TABLES AND EXPOSURE-RESPONSE ARRAYS	A-l
    A.I. Data Extraction: Preparation of Preliminary Evidence Tables and Exposure-Response
       Arrays for Primary Studies	A-l
    A.2. Effects in Humans	A-2
    A.3. Effects in Animals	A-6
       A.3.1. Thyroid Effects Evidence Table and Exposure-response Array	A-6
       A.3.2. Liver Effects Evidence Table and Exposure-response Array	A-13
       A.3.3. Neurological  Effects Evidence Table and Exposure-response Array	A-19
       A.3.4. Developmental Effects Evidence Table and Exposure-response Array	A-22
       A.3.5. Reproductive Effects Evidence Table and Exposure-response Array	A-25
       A.3.6. Immune Effects Evidence Table and Exposure-response Array	A-29
       A.3.7. Information on test material used in experimental animal studies	A-33
APPENDIX B. PRELIMINARY MECHANISTIC STUDY INFORMATION	B-l
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                              Preliminary Materials for the IRIS Toxicological Review ofHBCD
TABLES
Table 2-1. Summary of detailed search strategies for HBCD	2-4
Table 2-2. Processes used to augment the search of core databases for HBCD	2-5
Table 3-1. General and outcome-specific considerations for HBCD human study evaluation	3-8
Table 3-2. Questions and relevant experimental information for evaluation of experimental
              animal studies	3-9
Table A-l. Evidence pertaining to effects in humans	A-2
Table A-2. Evidence pertaining to thyroid effects in animals following oral exposure to HBCD	A-6
Table A-3. Evidence pertaining to liver effects in animals following oral exposure to HBCD	A-13
Table A-4. Evidence pertaining to neurological effects in animals following oral exposure to
              HBCD	A-19
Table A-5. Evidence pertaining to developmental effects in animals following oral exposure to
              HBCD	A-22
Table A-6. Evidence pertaining to reproductive effects in animals following oral exposure to
              HBCD	A-25
Table A-7. Evidence pertaining to immune effects in animals following oral exposure to HBCD	A-29
Table A-8. Test material information	A-33
Table B-l. HBCD mechanistic studies	B-2
FIGURES
Figure 2-1. Summary of literature search and screening process for HBCD	2-3
Figure A-l. Exposure-response array of thyroid effects following oral exposure to HBCD	A-12
Figure A-2. Exposure-response array of liver effects following oral exposure to HBCD	A-18
Figure A-3. Exposure-response array of neurological effects following oral exposure to HBCD	A-21
Figure A-4. Exposure-response array of developmental effects following oral exposure to HBCD	A-24
Figure A-5. Exposure-response array of reproductive effects following oral exposure to HBCD	A-28
Figure A-6. Exposure-response array of immune effects following oral exposure to  HBCD	A-32
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
 1
 2    PREFACE
 3          This draft document presents a planning and scoping summary, information on the
 4    approaches used to identify pertinent literature and primary studies, results of the literature
 5    search, approaches for selection of studies for hazard identification, presentation of characteristics
 6    and information from primary studies in evidence tables and exposure-response arrays, and
 7    mechanistic information in a summary table for hexabromocyclododecane (henceforth referred to
 8    as HBCD) prepared under the auspices of EPA's Integrated Risk Information System (IRIS)
 9    Program. This material is being released for public viewing and comment prior to a public meeting,
10    providing an opportunity for the IRIS Program to engage in early discussions with stakeholders and
11    the public on data that may be used to identify adverse health effects and characterize dose-
12    response relationships.
13          The planning and scoping summary includes information on the uses of HBCD, occurrence
14    ofHBCD in the environment, and the rationale and scope for the development of the assessment
15    This information is responsive to recommendations in the 2009 National Research Council (NRC)
16    report Science and Decisions: Advancing Risk Assessment (NRC. 2009] related to planning and
17    scoping in the risk assessment process.
18          The preliminary materials are also responsive to the NRC 2011 report Review of the
19    Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde (NRC, 2011]. The IRIS
20    Program's implementation of the NRC recommendations is following a phased approach that is
21    consistent with the NRC's "Roadmap for Revision" as described in Chapter 7 of the formaldehyde
22    review report. The NRC stated that "the committee recognizes that the changes suggested would
23    involve a multi-year process and extensive effort by the staff at the National Center for
24    Environmental Assessment and input and review by the EPA Science Advisory Board and others."
25    Phase 1 of implementation has focused on a subset of the short-term recommendations, such as
26    editing and streamlining documents, increasing transparency and clarity, and using more tables,
27    figures, and appendices to present information and data in assessments. Phase 1 also focused on
28    assessments near the end of the development process and close to final posting.  Phase 2 of
29    implementation is focused on assessments that are in the beginning stages of assessment
30    development The IRIS HBCD assessment is in Phase 2 and represents a significant advancement in
31    implementing the NRC recommendations. In the development of this assessment many of the
32    recommendations are being implemented in full, while others are being implemented in part.
33    Achieving full and robust implementation of certain recommendations will be an evolving process
34    with input and feedback from the public, stakeholders, and independent external peer review.
35    Phase 3 of implementation will incorporate the longer-term recommendations made by the NRC,
36    including the development of a standardized approach to describe the strength of evidence for
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    noncancer effects. On May 16, 2012, EPA announced1 that as a part of a review of the IRIS
 2    Program's assessment development process, the NRC will also review current methods for weight-
 3    of-evidence analyses and recommend approaches for weighing scientific evidence for chemical
 4    hazard identification. This effort is included in Phase 3 of EPA's implementation plan.
 5          The literature search strategy, which describes the processes for identifying scientific
 6    literature, screening studies for consideration, and identifying primary sources of health effects
 7    data, is responsive to NRC recommendations regarding the development of a systematic and
 8    transparent approach for identifying the primary literature for analysis. The preliminary materials
 9    also describe EPA's approach for the selection of primary studies to be included in the evidence
10    tables, as well as the approach for evaluating methodological features of studies that will be
11    considered in the overall evaluation and synthesis of evidence for each health effect  The
12    development of these materials is in response to the NRC recommendation to thoroughly evaluate
13    critical studies with standardized approaches that are formulated and based on the type of research
14    (e.g., observational epidemiology or animal bioassays). In addition, NRC recommendations for
15    standardized presentation of key study data are addressed by the development of the preliminary
16    evidence tables and preliminary exposure-response arrays for primary health effect information,
17    and summary tables for mechanistic data.
18          EPA welcomes all comments on the preliminary materials in this document, including the
19    following:
20          •  the clarity and transparency of the materials;
21          •  the approach for identifying pertinent studies;
22          •  the selection of primary studies for data extraction to preliminary evidence tables and
23              exposure-response arrays;
24          •  any methodological considerations that could affect the interpretation of or confidence
25              in study results; and
26          •  any additional studies published or nearing publication that may provide data for the
27              evaluation of human health hazard or dose-response relationships.
28          The preliminary evidence tables and exposure-response  arrays should be regarded solely as
29    representing the data on each endpoint that have been identified as a result of the draft literature
30    search strategy. They do not reflect any conclusions as to hazard identification or dose-response
31    assessment.
32          After obtaining public input and conducting additional study evaluation and data
33    integration, EPA will revise these materials to support the hazard identification and dose-response
34    assessment in a draft Toxicological Review that will be made available for public comment
      1 EPA Announces NAS' Review of IRIS Assessment Development Process.  05/16/2012.
      http://yosemite.epa.gOV/opa/admpress.nsf/0/lce2a7875daf093485257a000054df5470penDocument

                This document is a draft for review purposes only and does not constitute Agency policy.
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
      1.   PLANNING  AND SCOPING  SUMMARY
 1    1.1.  HBCD Chemistry and Uses
 2          Flame retardant chemicals are used in a variety of products to reduce fire risks. HBCD has
 3    been used to treat multiple products, including textiles, polystyrene thermal insulation and certain
 4    polystyrene plastic used for electronic devices, and others. In November of 2014, the listing of
 5    HBCD as a Persistent Organic Pollutant (POP) under the Stockholm Convention will take effect, and
 6    the allowable uses of HBCD will be restricted. Uses in polystyrene insulation will be allowed to
 7    continue under the Convention until 2019 when alternatives are expected to be available for this
 8    use.2 In 2012, EPA proposed a rule under the Toxic Substances Control Act to restrict uses of
 9    HBCD.3
10          Previous uses of HBCD, such as on textiles, may have led to greater human exposure than
11    insulation uses.4 In polystyrene insulation, HBCD is added to the polymer at high temperature
12    and/or pressure. The material is not chemically bound to the polystyrene, however its diffusion
13    from insulation foam is expected to be low.5  Although HBCD releases to the human environment
14    may be attenuated through changes in use, hundreds of millions of pounds of the material have
15    been used and are present in treated products at varying  lifecycle stages.  Given continued HBCD
16    use and the persistence of the chemical, the potential for human exposure is likely to continue for
17    many years.
18          HBCD is not a naturally occurring chemical. The technical product is generally reported to
19    exceed 94% purity with detected impurities including tetrabromocyclododecane and part per
20    billion levels of polybrominated dibenzofurans.6 The chemical HBCD can exist in 16 isomeric
21    forms.  It may be designated as a mixture of allisomers (CASRN 25637-99-4) or as a mixture of
22    three main diastereomers when the bromine atoms are in the 1, 2, 5, 6, 9 and 10 positions (CASRN
23    3194-55-6). Commercial HBCD contains primarily a mixture of the three  major diastereomers,
24    termed a, (3andy. Four commercial grades of HBCD have been used and vary in proportions of the
25    a, p and y diastereomers: low melt, medium range, high melt and thermally stabilized.7
26
      2 http://cen.acs.org/articles/91/il9/Global-Ban-Flame-Retardant.html
      3 http://www.reginfo.gov/public/do/eAgendaViewRule?pubId=201310&RIN=2070-AI88
      4 http://www.ec.gc. ca/ese-ees/default.asp?lang=En&n=7882C148-l#a3
      5 http://www.epa.gov/dfe/pubs/projects/hbcd/hbcd-draft-full-report.pdf
      6 European Commission Risk Assessment for Hexabromocyclododecane
      [http://esis.jrc.ec.europa.eu/doc/risk assessment/REPORT/hbcddreport044.pdf]
      7 Environment Canada Screening Assessment Report for Hexabromocyclododecane
      [http://www.ec.gc.ca/ese-ees/default.asp?lang=En&n=7882C148-l#al1

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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
                                                            Br
                                         Br
 1                                              Br
 2                             1,2,5,6,9,10-Hexabromocyclododecane
 3                                 (CizHisBre; CASRN 3194-55-6}
 4

 5    1.2. HBCD in the Environment
 6          In water and soil, HBCD is generally understood to degrade slowly. Environmental half life
 7    estimates from field studies suggest half lives exceeding one year. HBCD has low water solubility
 8    and binds to sediment or suspended solids in aquatic environments. In soil, it binds strongly to soil
 9    organic matter, which reduces the amount that can leach into groundwater. Because it is
10    semivolatile, HBCD exposed to air can partition into the atmosphere. The volatility and
11    environmental persistence of HBCD account for its detection far from use and waste sites (i.e., the
12    arctic).
13          In ecosystems, HBCD is reported to bioconcentrate and biomagnify.  Consistent with HBCD's
14    reported potential to bioaccumulate, the chemical is on several state and international priority
15    lists8:
16       •  the Canadian  Environmental Protection Act Environmental Registry Domestic Substances
17          List as Persistent, Bioaccumulative, and Inherently Toxic;
18       •  Washington Department of Ecology's Persistent, Bioaccumulative, Toxic Chemicals in
19          Washington State's Administrative Code;
20       •  the European Commission as persistent, bioaccumulative and toxic in the candidate list of
21          Substances of Very High Concern;
22       •  the California Environmental Contaminant Biomonitoring Program as a Priority Chemical
23       •  the Stockholm Convention as a Persistent Organic Pollutant
24
25          HBCD has been detected in human breast milk, adipose tissue and blood. HBCD was
26    measured in fetal liver tissue at concentrations ranging from below the detection limit to
27    4,500 ng/g of lipid.9 In homes, HBCD has been detected in air and dust Concentrations reported in
      8 http://www.dtsc.ca.gov/SCP/upload/Group-Member-Candidate-Chemicals-List.pdf
      9 http://www.sciencedirect.com/science/article/pii/S0048969713009285tf

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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    fish have been as high as 8,000 ng/g of lipid but most reported levels are below 50 ng/g of lipid.  An
 2    estimate of human dietary intake in the U.S. is reported to be 15.4 ng/day.10
 3          A 2008 European risk assessment found that exposure to indoor air and airborne dust in
 4    homes was an insignificant route of exposure and focused more on dietary exposure.n However,
 5    other researchers have suggested that while diet is an important route of exposure, inhalation and
 6    ingestion of dust are increasingly being considered to be the major sources of human exposure.12
 7          Release to the environment during the manufacturing process is considered to be low.13  To
 8    reduce releases of HBCD during the manufacturing process, some facilities have put in place dust
 9    filtering systems, catalytic burning systems, wastewater treatment systems involving activated
10    carbon and/or biomembrane reactors, and specialized waste incineration processes.14

11    1.3. Rationale for the Development  of the Toxicological Review
12          Given its potential for widespread human exposure, the IRIS Program is developing an
13    assessment of HBCD to address multiple needs.  Several activities that would benefit from the IRIS
14    assessment of HBCD are presented below:
15       •  Due to concerns associated with HBCD exposure and toxicity, EPA has identified HBCD as a
16          priority and released a detailed Action Plan announcing several rulemakings being
17          considered under the Toxic Substances Control Act and the Toxics Release Inventory.15 An
18          HBCD IRIS assessment would provide useful information for rulemaking and HBCD health
19          risk assessment.
20       •  EPA is also reviewing alternatives for major HBCD uses through the Design for the
21          Environment Program (DfE).16 DfE evaluates chemicals' lifecycles and assesses risks of
22          potential replacement chemicals to identify feasible alternatives. An IRIS assessment of
23          HBCD could help inform the DfE comparative toxicity and risk assessment for identifying
24          safer alternatives. The information developed by DfE could encourage the use of safer
25          chemicals and technologies.17
26       •  EPA, the Food and Drug Administration (FDA) and states issue advice about consuming fish
27          that may be unhealthy to eat due to contamination.  IRIS assessments provide useful
28          information on chemicals' toxicity for developing these advisories, and EPA guidance for
29          developing fish advisories recommends that IRIS values be used in setting screening values
      10 http:// www.ec.gc. ca/ese-ees/default.asp?lang=En&n=A167D02F-l#all
      11 http://esis.jrc.ec.europa.eu/doc/risk assessment/REPORT/hbcddreport044.pdf
      12 http://www.sciencedirect.com/science/article/pii/S0048969713009285tf
      13 http://echa.europa.eu/documents/10162/13640/tech rep hbcdd en.pdf
      14 http://www.bsef.com/uploads/Factsheet HBCD 25-10-2012.pdf
      15 http://www.epa.gov/oppt/existingchemicals/pubs/actionplans/hbcd.htmltfaddress
      16 http://www.epa.gov/dfe/pubs/projects/hbcd/about.htm
      17 http://www.epa.gov/oppt/existingchemicals/pubs/actionplans/hbcd.htmltfaddress

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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1          for consumption levels.18 Because HBCD has been identified by several organizations as a
 2          toxic, persistent and bioaccumulating chemical, an IRIS assessment may inform whether
 3          advisories are warranted.
 4       •  HBCD was considered for inclusion on the third Contaminant Candidate List (CCL 3) under
 5          the Safe Drinking Water Act19 but it was not included. EPA is required to update this list of
 6          water contaminants every five years and identify those contaminants that may warrant
 7          future regulatory action. EPA uses a multi-step process to evaluate occurrence and health
 8          information to determine the substances that are included on the CCL. IRIS Reference
 9          Values, cancer dose-response information and cancer descriptors, when they are available,
10          are used to evaluate health effects of potential CCL chemicals.
11       •  IRIS values are also used in the development of Human Health Ambient Water Quality
12          Criteria (HH-AWQC) under the Clean Water Act A HH-AWQC is the highest concentration of
13          a pollutant in water that is not expected to pose a significant risk to human health when
14          considering ingestion of water and aquatic organisms or aquatic organisms only. These
15          values are used by states in controlling discharges to ambient water bodies with "drinkable
16          fishable" use designations.
17       •  Given HBCD's level of use and its environmental persistence, an IRIS assessment is
18          anticipated to be useful for EPA programs involved in waste management and site cleanup.
19       •  HBCD has been identified as a Substance of Very High Concern (SVHC) under the European
20          Registration, Evaluation, Authorisation and Restriction of Chemicals Program (REACH). As
21          an SVHC, HBCD may become subject to the "authorisation" process to ensure less dangerous
22          substances are used in HBCD replacement

23    1.4. General Scope of the Toxicological Review
24          The Toxicological Review of HBCD will consider health effects data for cancer and
25    noncancer endpoints from subchronic and chronic exposures to HBCD. Three broad types of
26    studies, if available, will be used to inform human health effects: controlled human exposure,
27    epidemiologic, and experimental studies. Mechanistic or mode of action data will be evaluated and
28    may inform questions of human relevance, susceptibility, and dose-response relationships.
29    Considering the potential uses of IRIS information and potential pathways of exposure, an IRIS
30    assessment of HBCD would be expected to incorporate the following, provided that adequate data
31    are available:
32       •  Systematic identification of hazards from long-term exposures
33       •  Analysis of mode of action information, if available
      18
      http://water.epa.gov/scitech/swguidance/fishshellfish/techguidance/risk/upload/2009 04 23 fish advice
      volumel vlcover.pdf
      19 http://water.epa.gov/scitech/drinkingwater/dws/ccl/upload/CCL3 Chemicals Universe 08-31-
      09 508 v3.pdf

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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1       •   Dose-response relationships for identified hazards
 2       •   Chronic Reference Concentration (RfC)
 3       •   Chronic Reference Dose (RfD)
 4       •   Cancer assessment and weight of evidence descriptor for oral and inhalation exposure,
 5           including dose-response information
 6       •   Identification of human populations and developmental stages with potentially greater
 7           susceptibility to HBCD
 8
 9           The HBCD assessment will rely on existing analytical tools and toxicity data and contain
10    qualitative characterizations of uncertainty and variability related to hazard assessment and dose-
11    response relationships. The development process for this assessment will provide opportunities
12    for public comment and dialogue and includes independent external peer review.
13
14
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD


      2.   DRAFT LITERATURE SEARCH AND SCREENING
           STRATEGY
 1          The NRC [NRG, 2011] recommended that EPA develop a detailed search strategy utilizing a
 2    graphical display documenting how initial search findings are narrowed to the final studies that are
 3    selected for further evaluation on the basis of inclusion and exclusion criteria.  Following these
 4    recommendations, a literature search and screening strategy was applied to identify literature
 5    related to characterizing the health effects of HBCD. This strategy consisted of a search of online
 6    scientific databases and other sources, casting a wide net in order to identify all potentially
 7    pertinent studies. In subsequent steps, references were screened to exclude papers not pertinent
 8    to an assessment of the health effects ofHBCD, and remaining references were sorted into
 9    categories for further evaluation.
10          The literature search for HBCD was conducted in four online scientific databases, including
11    PubMed, Toxline, Toxcenter, and TSCATS, in August 2013. The detailed search approach, including
12    the search strings and number of citations identified per database, is presented in Table 2-1. This
13    search of online databases identified 635 citations (after electronically eliminating duplicates). The
14    computerized database searches were also supplemented by a review of online regulatory sources
15    as well as "forward" and "backward" searches of Web of Science using several key references (Table
16    2-2); 29 citations were obtained using these additional search strategies. In total, 664 citations
17    were identified using online scientific databases and additional search strategies.
18          These citations were screened using the title, abstract, and in limited instances, full text for
19    pertinence to examining the health effects of HBCD exposure. The process for screening the
20    literature is described below and is shown graphically in Figure 2-1.
21          •  41 references were identified as potential primary sources of health effects data and
22             were considered for data extraction to evidence tables and exposure-response arrays.
23          •  118 references were identified as not being pertinent and were excluded from further
24             consideration (see Figure 2-1 for exclusion categories).
25          •  39 references were kept for further review. This category includes references that did
26             not provide enough material to evaluate pertinence (e.g., abstract not available).
27          •  357 references were identified as not primary sources of health effects data (e.g.,
28             reviews and studies with chemical/physical property information), but were kept as
29             additional resources for development of the Toxicological Review.
30          •  109 studies were identified as supporting studies; these included 54 studies providing
31             genotoxicity and other mechanistic information and 55 toxicokinetic studies. The 54
          This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD


1              supporting studies with genotoxicity and other mechanistic information were
2              considered for inclusion in a compendium of mechanistic study information.
3           The literature will be regularly monitored for the publication of new studies and a formal
4    updated literature search and screen will be conducted after the IRIS bimonthly public meeting
5    discussing these preliminary materials.
6           The documentation and results for the literature search and screen can be found on the
7    Health and Environmental Research Online (HERO) website (http://hero.epa.gov/HBCD).20
     20 HERO (Health and Environmental Research On-line) is a database of scientific studies and other references
     used to develop EPA's risk assessments aimed at understanding the health and environmental effects of
     pollutants and chemicals. It is developed and managed in EPA's Office of Research and Development (ORD)
     by the National Center for Environmental Assessment (NCEA). The database includes more than 300,000
     scientific articles from the peer-reviewed literature. New studies are added continuously to HERO.

     It is important to note that the HERO database will be regularly updated as additional references are
     identified during assessment development. Therefore, the numbers of references (by tag) displayed on the
     HERO webpage for HBCD may not match the numbers of references identified in Figure 2-1 (current through
     March 2 014).

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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
                      Database Searches (see Table 2-1 for keywords and limits)
             Pubmed
              n=468
                       Toxline
                        n=29
Web of Science
    n=326
TSCATS 2
  n=65
                            n=635 (after duplicates removed electronically)
         Additional Search Strategies
        (see Table 2-2 for methods and results)
                     n=29
                                        Combined Dataset
                                              n=664
                                  Manual Screening for Pertinence
                                      (Title/Abstract/Full Text)
1
2
      Excluded (n=118)
      3 Abstract only
      52 Not chemical specific
      42 Measurement methods
      14 Chemical treatment/disposal/remediation
      7 Miscellaneous
                                                       Kept for Further Review (n=39)
                                                       30 No abstract
                                                       2 Inadequate reporting in abstract
                                                       7 Foreign language
                                           Kept as Additional Resource (n=357)
                                           18 Regulatory documents
                                           41 Reviews
                                           14 Risk assessments
                                           22 Chemical/physical properties
                                           33 Fate and transport
                                           70 Exposure levels
                                           153 Ecosystem effects
                                           6 Mixtures only
                                                       Supporting Studies (n=109)
                                                       54 Mechanistic and genotoxicity studies
                                                       55 Toxicokinetics
                                                       Primary Source of Health Effects Data
                                                       (n=41)
                                                       7 Human health effect studies
                                                       34 Animal toxicology studies
Figure 2-1. Summary of literature search and screening process for HBCD.
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                       Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Table 2-1. Summary of detailed search strategies for HBCD
Database
Search Date
PubMed
08/20/13

Web of Science
08/21/13

































ToxLine
08/22/13



TSCATS 1
08/22/13
TSCATS 2

Set#
1A


1B1


































1C1

1C2


1D1
1D2
1E1

Terms
hexabromocyclododecane[nm] OR "3194-55-6"[tw] OR "25637-99-4" [tw]
OR "l,2,5,6,9,10-hexabromocyclodecane"[tw] OR
hexabromocyclododecane*[tw] OR hbcd[tw] OR hbcds[tw]
(TS="l,2,5,6,9,10-hexabromocyclodecane" OR
TS="hexabromocyclododecane" ORTS=hexabromocyclododecane* OR
TS="HBCD" OR TS="HBCDs") AND ((WC=("Toxicology" OR "Endocrinology
& Metabolism" OR "Gastroenterology & Hepatology" OR
"Gastroenterology & Hepatology" OR "Hematology" OR "Neurosciences"
OR "Obstetrics & Gynecology" OR "Pharmacology & Pharmacy" OR
"Physiology" OR "Respiratory System" OR "Urology & Nephrology" OR
"Anatomy & Morphology" OR "Andrology" OR "Pathology" OR
"Otorhinolaryngology" OR "Ophthalmology" OR "Pediatrics" OR
"Oncology" OR "Reproductive Biology" OR "Developmental Biology" OR
"Biology" OR "Dermatology" OR "Allergy" OR "Public, Environmental &
Occupational Health") OR SU=("Anatomy & Morphology" OR
"Cardiovascular System & Cardiology" OR "Developmental Biology" OR
"Endocrinology & Metabolism" OR "Gastroenterology & Hepatology" OR
"Hematology" OR "Immunology" OR "Neurosciences & Neurology" OR
"Obstetrics & Gynecology" OR "Oncology" OR "Ophthalmology" OR
"Pathology" OR "Pediatrics" OR "Pharmacology & Pharmacy" OR
"Physiology" OR "Public, Environmental & Occupational Health" OR
"Respiratory System" OR "Toxicology" OR "Urology & Nephrology" OR
"Reproductive Biology" OR "Dermatology" OR "Allergy")) OR
(WC="veterinary sciences" AND (TS="rat" OR TS="rats" OR TS="mouse"
ORTS="murine" ORTS="mice" ORTS="guinea" ORTS="muridae" OR
TS=rabbit* ORTS=lagomorph* ORTS=hamster* ORTS=ferret* OR
TS=gerbil* ORTS=rodent* ORTS="dog" ORTS="dogs" ORTS=beagle* OR
TS="canine" ORTS="cats" ORTS="feline" ORTS="pig" ORTS="pigs" OR
TS="swine" ORTS="porcine" ORTS=monkey* ORTS=macaque* OR
TS=baboon* ORTS=marmoset*)) OR (TS=toxic* AND (TS="rat" OR
TS="rats" ORTS="mouse" ORTS="murine" ORTS="mice" ORTS="guinea"
ORTS="muridae"ORTS=rabbit* ORTS=lagomorph* ORTS=hamster* OR
TS=ferret* ORTS=gerbil* ORTS=rodent* ORTS="dog" ORTS="dogs" OR
TS=beagle* ORTS="canine" ORTS="cats" ORTS="feline" ORTS="pig" OR
TS="pigs" ORTS="swine" ORTS="porcine" ORTS=monkey* OR
TS=macaque* ORTS=baboon* ORTS=marmoset* ORTS="child" OR
TS="children" ORTS=adolescen* ORTS=infant* ORTS="WORKER" OR
TS="HUMAN" ORTS=patient*)) ORTS="exposure")
@OR+(@term+@rn+25637-99-4+@term+@rn+3194-55-
6)+@NOT+@org+pubmed+pubdart+"nih+reporter"+tscats
@OR+("hexabromocyclodecane"+"hexabromocyclododecane"+"hexabro
mocyclododecane"+"hexabromocyclododecanes"+"hbcd"+"hbcds")+@N
OT+@org+pubmed+pubdart+"nih+reporter"+tscats
@term+@rn+25637-99-4+@AND+@org+tscats
@term+@rn+3194-55-6+@and+@org+tscats
3194-55-6, 25637-99-4

Hits
468


326


































22

20


12
53
10
This document is a preliminary draft for review purposes only and does not constitute Agency policy,
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
08/22/13
TSCA 8e/FYI
recent
submissions
08/22/13
Merged
Reference Set

1E1
1
date limited, 2000-date of search
Google: 3194-55-6, 25637-99-4 with (8e or fyi) tsca
(duplicates eliminated through electronic screen)

4
635
1

2
Table 2-2. Processes used to augment the search of core databases for HBCD

System Used
Manual search of
citations from
regulatory documents





Web of Science,
forward search





















Web of Science,
backward search



Selected Key Reference(s) or Sources
European Commission. (2008). Risk Assessment:
Hexabromocyclododecane. Final report.
Luxembourg: Office for Official Publications of the
European Communities
Environment Canada. (2011). Screening Assessment
Report on Hexabromocyclododecane; Chemical
Abstracts Service Registry Number 3194-55-6,
Environment Canada, Health Canada
Ema, M; Fujii, S; Hirata-Koizumi, M; Matsumoto, M.
(2008). Two-generation reproductive toxicity study of
the flame retardant hexabromocyclododecane in
rats. Reprod Toxicol 25: 335-351
Eriksson, P; Fischer, C; Wallin, M; Jakobsson, E;
Fredriksson, A. (2006). Impaired behaviour, learning
and memory, in adult mice neonatally exposed to
hexabromocyclododecane (HBCDD). Environ Toxicol
Pharmacol 21: 317-322
Saegusa, Y; Fujimoto, H; Woo, GH; Inoue, K;
Takahashi, M; Mitsumori, K; Hirose, M; Nishikawa, A;
Shibutani, M. (2009). Developmental toxicity of
brominated flame retardants, tetrabromobisphenol A
and 1,2,5,6,9,10-hexabromocyclododecane, in rat
offspring after maternal exposure from mid-gestation
through lactation. Reprod Toxicol 28: 456-467
van Der Ven, LTM; van De Kuil, T; Leonards, PEG;
Slob, W; Lilienthal, H; Litens, S; Herlin, M; Hakansson,
H; Canton, RF; van Den Berg, M; Visser, TJ; van
Loveren, H; Vos, JG; Piersma, AH. (2009). Endocrine
effects of hexabromocyclododecane (HBCD) in a one-
generation reproduction study in Wistar rats. Toxicol
Lett 185: 51-62
Ema, M; Fujii, S; Hirata-Koizumi, M; Matsumoto, M.
(2008). Two-generation reproductive toxicity study of
the flame retardant hexabromocyclododecane in
rats. Reprod Toxicol 25: 335-351

Date
9/2013



9/2013



9/2013



9/2013




9/2013






9/2013






9/2013



Additional References
Identified
7 citations added



0 citations added



0 citations added



0 citations added




0 citations added






0 citations added






2 citations added



         This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                 2-5         DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
System Used


References obtained
during the
assessment process
Background Check









Selected Key Reference(s) or Sources
Eriksson, P; Fischer, C; Wallin, M; Jakobsson, E;
Fredriksson, A. (2006). Impaired behaviour, learning
and memory, in adult mice neonatally exposed to
hexabromocyclododecane (HBCDD). Environ Toxicol
Pharmacol 21: 317-322
Saegusa, Y; Fujimoto, H; Woo, GH; Inoue, K;
Takahashi, M; Mitsumori, K; Hirose, M; Nishikawa, A;
Shibutani, M. (2009). Developmental toxicity of
brominated flame retardants, tetrabromobisphenol A
and 1,2,5,6,9,10-hexabromocyclododecane, in rat
offspring after maternal exposure from mid-gestation
through lactation. Reprod Toxicol 28: 456-467
van Der Ven, LTM; van De Kuil, T; Leonards, PEG;
Slob, W; Lilienthal, H; Litens, S; Merlin, M; Hakansson,
H; Canton, RF; van Den Berg, M; Visser, TJ; van
Loveren, H; Vos, JG; Piersma, AH. (2009). Endocrine
effects of hexabromocyclododecane (HBCD) in a one-
generation reproduction study in Wistar rats. Toxicol
Lett 185: 51-62
Snowball search
Searched a combination of CASRNs and synonyms on
the following databases:
ACGIH (http://www.acgih.org/home.htm)
ATSDR
(httD://www.atsdr.cdc.gov/substances/index.asD)
CalEPA Office of Environmental Health Hazard
Assessment (http://www.oehha.ca.gov/risk.html)
Search this as well as the following sites (save the
first 50 results)
OEHHA Toxicity Criteria Database
(http://www.oehha.ca.gov/tcdb/index.asp)
Biomonitoring California-Priority Chemicals
(http://www.oehha.ca.gov/multimedia/biomon/pdf/
PriorityChemsCurrent.pdf)
Biomonitoring California-Designated Chemicals
(http://www.oehha.ca.gov/multimedia/biomon/pdf/
DesignatedChemCurrent.pdf)
Cal/Ecotox Database
(httD://www.oehha.ca.gov/scriDts/cal ecotox/CHEM
LIST. ASP)
OEHHA Fact Sheets
(http://www. oehha.ca.gov/Dublic info/facts/index. h
tml)
Non-cancer health effects Table (RELs)
http://www.oehha.ca.gov/air/allrels.html
and Cancer Potency Factors (Appendix A and
AppendixB)
Date
9/2013
9/2013
9/2013
9/2013
8/26/2013









Additional References
Identified
1 citation added
0 citations added
0 citations added
9 citations added
10 citations added









This document is a preliminary draft for review purposes only and does not constitute Agency policy,
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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
System Used

Selected Key Reference(s) or Sources
http://www.oehha.ca.gov/air/hot spots/tsd052909.
html
CPSC
(http://www.cpsc.gov)
eChemPortal (participating databases: ACToR,
AGRITOX, CCR, CCR DATA, CESAR, CHRIP, ECHA
CHEM, EnviChem, ESIS, GHS-J, HPVIS, HSDB, HSNO
CCID, INCHEM, J-CHECK, JECDB, NICNAS PEC, OECD
HPV, OECDSIDS IUCLID, SIDS UNEP, UKCCRMP
Outputs, US EPA IRIS, US EPA SRS)
(http://www.echemportal.org/echemportal/participa
nt/page.action?pagelD=9)
Environment Canada - Search entire site
(http://www.ee. gc.ca/default.asp?lang=En&n=ECD35
C36) if not found below:
Toxic Substances Managed Under CEPA
(http://www.ec.gc.ca/toxiques-
toxics/Default.asp?lang=En&n=98E80CC6-l) Search
results
Final Assessments (http://www.ec.gc.ca/lcpe-
cepa/default.asp?lang=En&xml=09F567A7-BlEE-
1FEE-73DB-8AE6C1EB7658)
Draft Assessments (http://www.ec.gc.ca/lcpe-
cepa/default.asp?lang=En&xml=6892C255-5597-
C162-95FC-4B905320F8C9)
EPA Acute Exposure Guideline Levels
(http://www.epa.gov/oppt/aegl/pubs/chemlist.htm)
EPA - IRISTrack/New Assessments and Reviews
(http://cfpub.epa.gov/ncea/iristrac/) to find dates
(http://www.epa.gov/ncea/iris/index.html) to find
data
EPA NSCEP
(http://www.epa.gov/ncepihom/)
EPA Science Inventory
(http://cfpub.epa.gov/si/)
FDA
(http://www.fda.gov/)
Federal Docket
(www.regulations.gov)
Health Canada First Priority List Assessments
(http://www.hc-sc.gc.ca/ewh-
semt/pubs/contaminants/psll-lspl/index-eng.php)
Health Canada Second Priority List Assessments
(http://www.hc-sc.gc.ca/ewh-
semt/pubs/contaminants/ps!2-lsp2/index-eng.php)
IARC
Index:
http://monographs.iarc.fr/ENG/Monographs/voll01/
monol01-B02-B03.pdf
NAP -Search Site
Date

Additional References
Identified

This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                         2-7         DRAFT—DO NOT CITE OR QUOTE

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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
System Used

Selected Key Reference(s) or Sources
(http://www.nap.edu/)
NCI
(http://www.cancer.gov)
NCTR
(http://www.fda.gov/AboutFDA/CentersOffices/OC/
OfficeofScientificand Medical Programs/NCTR/default.
htm)
NIEHS
http://www.niehs.nih.gov/
NICNAS (PEC only covered by eChemPortal)
(http://www.nicnas.gov.au/industry/aics/search.asp)
NIOSH
(http://www.cdc.gov/niosh/topics/)
NIOSHTIC2
(http://www2a.cdc.gov/nioshtic-2/)
NTP - RoC, status, results, and management reports
12th Report On Carcinogens:
(http://ntp.niehs.nih. gov/?obiectid=03C9AF75-E!BF-
FF40-DBA9EC0928DF8B15 )
NTP Site Search:
http://ntpsearch.niehs.nih. gov/texis/search/?query=
arsenic&pr=ntp web entire site all&mu=Entire+NT
P+Site
OS HA
(http://www.osha.gov/dts/chemicalsampling/toc/toc
chemsamp.html)
RTECS
http://www.ccohs.ca/search.html
Date

Additional References
Identified

1
2
          This document is a preliminary draft for review purposes only and does not constitute Agency policy,
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD


 i    3.  SELECTION OF  STUDIES  FOR HAZARD
 2        IDENTIFICATION
 3    3.1.  General Approach
 4          The NRC [NRG, 2011] recommended that after studies are identified for review by utilizing
 5    a transparent search strategy, the next step is to summarize the details and findings of the most
 6    pertinent studies in evidence tables. The NRC suggested that such tables should provide a link to
 7    the references, and include details of the study population and methods and key findings. This
 8    approach provides for a systematic and concise presentation of the evidence. The NRC also
 9    recommended that the methods and findings should then be evaluated with a standardized
10    approach. The approach that was outlined identified standard issues for the evaluation of
11    epidemiological and experimental animal studies.
12          In response to the NRC recommendations, each study retained after the literature search
13    and screen is evaluated for aspects of its design or conduct that could affect the interpretation of
14    results and the overall contribution to the synthesis of evidence for determination of hazard
15    potential. Much of the key information for conducting this evaluation can generally be found in the
16    study's methods section and in how the study results are reported. Importantly, this evaluation
17    does not consider study results or more specifically, the direction or magnitude of any reported
18    effects. For example, standard issues for evaluation of experimental animal data identified by the
19    NRC and adopted in this approach include consideration of the species and sex of animals studied,
20    dosing information (dose spacing, dose duration, and route of exposure), endpoints considered, and
21    the relevance of the endpoints to the human endpoints of concern.
22          To facilitate the evaluation outlined above, evidence tables are constructed that consistently
23    summarize the important information from each study in a standardized tabular format as
24    recommended by the NRC [NRC, 2011]. In general, the evidence tables include all studies that
25    inform the overall synthesis of evidence for hazard potential. At this stage, exclusion of studies may
26    unnecessarily narrow subsequent analyses by eliminating information that might later prove
27    useful. Premature exclusion might also give a false sense of the consistency of results across the
28    database of studies by unknowingly reducing the diversity of study results. Thus,  at this early stage
29    of study evaluation the goal is to be inclusive.
30          Even at this early stage, however, a study can be excluded if flaws in  its design or conduct
31    are so great that the results would not be considered credible. Such study design flaws are
32    discussed in a number of EPA's guidelines (see http://www.epa.gov/iris/backgrd.html] or
          This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    summarized in the draft Preamble to the IRIS Toxicological Review ("Preamble")21. Examples of
 2    these flaws include studies where impurities in the test chemical are so great as to prohibit
 3    attribution of the results to the chemical, or studies where concurrent or essential historical control
 4    information is lacking. Studies excluded because of fundamental flaws in their design or conduct
 5    are not included in evidence tables.  Instead, text accompanying the evidence tables lists the
 6    reasons that studies were excluded.
 7           The size of the database can influence both the type and number of evaluation criteria that
 8    are applied at this early stage. For example, if there are few studies on a health effect, additional
 9    evaluation criteria might not be needed, and thus the evidence tables may include all studies
10    without severe flaws. Especially with smaller databases, it is important to consider all studies and
11    not exclude studies unnecessarily. On the other hand, if there are many studies on a health effect
12    (e.g., more than 20), additional criteria could facilitate a more efficient review of the database and
13    help to focus on the more pertinent or stronger studies indicating the potential for hazard.  These
14    criteria could be specific to each type of study or a particular endpoint, and may consider factors
15    such as those discussed in EPA's guidelines or summarized in the draft Preamble. Application of
16    such additional criteria could result in initially setting aside some studies and not summarizing
17    them in the evidence tables.  Also, there may be situations in which the initial review of the
18    available data will lead to a decision to focus on a particular set of health effects, and to
19    exclude others  from further evaluation. This situation could occur, for example, with a chemical
20    with a large database with a few well-developed areas of research, but many other areas that
21    consist of sparse data offering a very limited basis for drawing conclusions regarding hazard. In
22    this case, EPA will focus on the more developed areas of research for hazard identification.

23    3.2. Selection of Primary Studies for Evidence Tables for HBCD

24    3.2.1.  Epidemiologic Studies
25           The initial review of epidemiologic studies was conducted for those that were retained after
26    the literature was manually screened for pertinence (title, abstract, and/or full text) (Figure 2-1;
27    Primary Sources of Health Effects Data). Five epidemiologic studies examined associations
28    between HBCD exposure and certain endocrine (including thyroid and reproductive hormone),
29    neurobehavioral, and developmental outcomes. None of these studies had severe flaws that would
30    compromise the credibility of their results. Because there are relatively few epidemiological
31    studies of HBCD, these studies are all included in the preliminary evidence tables.
32           Two human studies were not summarized in the evidence tables. One study examined bone
33    density as an outcome measure (Weiss et al., 2006), but no association with measures of HBCD
      21 See the draft Preamble in the Toxicological Review of Ammonia (revised external review draft) at
      http://cfpub.epa.gov/ncea/iris drafts/recordisplay.cfm?deid=254524 or in the Toxicological Review of
      Trimethylbenzenes (revised external review draft) at
      http://cfpub.epa.gov/ncea/iris drafts/recordisplay.cfrn?deid=254525.

           This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    exposure was observed and EPA is not further developing a review of this endpoint  A second
 2    study was a report of a human dermal patch test [McDonnell, 1972], a study design generally less
 3    pertinent for characterizing health hazards associated with chronic exposure. Nevertheless, these
 4    studies will still be considered as potential information sources during assessment development.

 5    3.2.2.  Experimental Animal Studies
 6          An initial review was also performed for the experimental animal studies identified in the
 7    literature search and screen (Figure 2-1; Primary Sources of Health Effects Data). The HBCD
 8    experimental animal database is relatively small, and consists of studies designed to  examine
 9    repeat-dose oral toxicity and specialized studies of reproductive and developmental  toxicity,
10    neurotoxicity and neurobehavioral toxicity, thyroid toxicity, and immunotoxicity. These studies are
11    pertinent to evaluating the health effects of HBCD associated with human environmental exposure,
12    and none had severe flaws that would compromise the credibility of their results. Because there
13    are relatively few experimental animal toxicity studies of HBCD, these studies are all included in the
14    preliminary evidence tables.
15          The HBCD experimental animal database also includes studies of acute toxicity and ocular
16    and dermal irritation. As these short-duration studies are generally less pertinent for
17    characterizing health hazards associated with chronic exposure, they are not summarized in the
18    preliminary evidence tables. Nevertheless, these studies will still be evaluated as possible sources
19    of toxicokinetic or mechanistic information during assessment development
20          The experimental database contains genotoxicity and other mechanistic studies that will
21    support the health assessment ofHBCD (see Figure 2-1; Supporting Studies).  Because mechanistic
22    studies are numerous and their designs are highly heterogeneous, extracting study design
23    information and results into evidence tables before identifying the health effects and potential
24    modes of action (MOAs) and/or adverse outcome pathways (AOPs) that are scientifically plausible
25    would be a resource intensive, yet potentially uninformative effort Instead, for this group of
26    studies, the preliminary materials provide a summary table of mechanistic studies (including
27    general information on the test system/assays, measured parameters, and the possible health
28    effect(s)  to which each mechanistic study may relate) as a useful starting point for future analysis of
29    support for possible MOAs/AOPs.

30    3.3. Preliminary Evidence Tables and Exposure-Response  Arrays
31          Data from the primary studies identified by the approaches outlined above have been
32    extracted and presented in evidence tables (Appendix A). The evidence tables present data from
33    studies related to a specific outcome or endpoint of toxicity.  At a minimum, the  evidence tables
34    include the relevant information for comparing key study characteristics such as study design,
35    exposure metrics, and dose-response information. Evidence tables will serve as an additional
36    method for presenting and evaluating the suitability of the data to inform hazard identification for
           This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    HBCD during the analysis of hazard potential and utility of the data for dose-response evaluation.
 2    The information in the preliminary evidence tables is also displayed graphically in preliminary
 3    exposure-response arrays. In these arrays, a significant effect (indicated by a filled circle) is based
 4    on statistical significance.
 5          A compendium of genotoxicity and other mechanistic studies that will support the HBCD
 6    health assessment, with general information on the test system used and endpoints evaluated, is
 7    presented in a mechanistic study summary table (Appendix B).
 8          The complete list of references considered in preparation of these materials can be found on
 9    the HERO website at http://hero.epa.gov/HBCD.

10    3.4. Study Characteristics That Will Be Considered in the Evaluation and
11         Synthesis of the Primary Studies for HBCD

12    3.4.1. Epidemiologic Studies
13          Several considerations will be used in EPA's evaluation of the studies of human health
14    effects ofHBCD. The general considerations for evaluating issues relating to the study population,
15    exposure, outcomes, confounding, and analysis are outlined in the draft Preamble. These, along
16    with more specific issues pertaining to exposure and outcomes studied, are described below and in
17    Table 3-1.
18
19    Study p op u lotion
20          The general considerations for evaluating issues relating to the study population include
21    adequate documentation of participant recruitment, including eligibility criteria, participation
22    rates, missing data, loss to follow-up, and general demographic characteristics.  This information is
23    used to evaluate the potential for selection bias, as well as to facilitate comparison of results across
24    different study populations. It is important to note that low participation rates, or even different
25    participation rates between exposed and non-exposed or between cases and controls, are not
26    evidence of selection bias. Rather,  selection bias arises from a differential pattern of participation
27    with respect to exposure and disease, e.g., if people with high exposure and the  outcome of interest
28    are more likely to participate than people with low exposure and the outcome.
29          The available epidemiological studies examined many different types of exposures
30    (brominated flame retardants as well as other types of compounds) within the context of research
31    on potential endocrine disrupters.  Individuals typically do not have knowledge of their exposure to
32    HBCD, and thus, knowledge of exposure or exposure level is unlikely to result in differential
33    participation with respect to outcomes. However, EPA will consider the possibility that a particular
34    concern about exposure to flame retardants would have motivated people to participate in a study
35    or to continue participation throughout a follow-up period.  EPA will also consider indirect ways in
36    which a common factor could contribute both to HBCD exposure and to a specific outcome. In the
           This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    absence of evidence that any of these scenarios is at play, EPA will not consider selection bias
 2    attributed to these factors to be a likely limitation of a study.
 3
 4    Exposure measures
 5           The general considerations for evaluating issues relating to exposure include
 6    characterization of exposure during the appropriate critical period for the outcomes under study,
 7    and use of appropriate ascertainment methods to classify individuals with regards to the exposure.
 8           There are some exposure-related issues specific to HBCD. The major sources of (non-
 9    occupational) exposure to HBCD are indoor dust and diet. HBCD can be measured in biological
10    samples; adipose tissue, serum and breast milk (which has a high proportion of lipids) are
11    preferred over urine or saliva because of the accumulation ofHBCD in fatty tissue and relatively
12    long half-life ofHBCD. The estimated half-life is likely on the order of weeks rather than days or
13    hours [Geyer etal., 2004], and thus in general a single spot measurement is not considered a
14    limitation for brominated flame retardants. However, HBCD levels could change more rapidly
15    during pregnancy and lactation, due to mobilization of maternal fat stores [Aurell and Cramer,
16    1966]: therefore EPA will consider these factors in evaluating the timing of sample collection in
17    relation to the critical window of exposure, if known, for the outcome (s] under study. Studies of
18    PBDEs in breast milk have shown relatively small variation in breast milk over time, with a 2-3%
19    decrease in PBDE concentration per month [Daniels etal.. 2010: Hooper etal.. 2007]: thus,
20    measurements of HBCD levels in breast milk are likely to  be a  good surrogate for infant post-natal
21    exposure, but less is known about the correlation with early periods of gestation. Measures of
22    HBCD in dust are likely to correlate well with concentrations in biological samples. One study by
23    Roosens etal. [2009] examined HBCD in serum and estimated HBCD ingestion from dust, and found
24    a high correlation of 0.86 between these two measures. This result is similar to or stronger than
25    correlations between other polybrominated flame retardant levels in dust and biomarker
26    measures, with correlations ranging from 0.3-0.8 [Stapletonetal.. 2012: Johnson etal.. 2010: Wu et
27    al.. 2007].
28           Measurement of HBCD in serum raises an additional issue with respect to the potential need
29    for adjustment for lipid levels, either through use of lipid  adjusted serum concentrations, or
30    inclusion of serum lipids as a covariate in multivariate analysis. Simulation studies indicate that the
31    former  approach (i.e., use of lipid adjusted concentrations] may lead to biased risk estimates
32    (Schistermanetal., 2005]. EPA will consider this potential bias in evaluating studies using lipid
33    adjusted concentrations.
34           HBCD comprises three isomers; a-HBCD appears to bioaccumulate more readily compared
35    to the other isomers, and may better reflect longer-term exposure. While some studies specify the
36    isomer  measured in biological samples (or state that all three  were measured together and
37    summed], others do not specify this information.  HBCD levels from these studies may represent a
38    'total' HBCD concentration, which is likely to be dominated by a-HBCD unless a significant exposure
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    event had occurred in the very recent past Thus, lack of specification of the isomer measured is
 2    unlikely to be a major limitation for epidemiology studies.
 3          Another issue with HBCD measured in either biological tissue or environmental media is the
 4    limit of detection (LOD) for the assay. A high proportion of samples below the LOD can reduce the
 5    ability of the study to evaluate associations, and particularly exposure-response patterns.
 6          EPA also considers the distribution of exposure in evaluating individual studies and
 7    comparing results among groups of studies. One consideration is the span of exposure levels (i.e.,
 8    the contrast between "high" and "low"): a study with a very narrow span may not have sufficient
 9    variability to detect an effect that would be seen over a broader range.  Another consideration is the
10    absolute level of exposure: different effect estimates may be expected in studies examining
11    different exposure levels.
12
13    Outcome measures
14          The general considerations for evaluating issues relating to outcomes include adequate
15    duration of exposure and follow-up in order to evaluate the outcomes of interest, and use of
16    appropriate ascertainment methods to classify individuals with regard to the outcome. The
17    primary outcomes examined in the epidemiology studies  are levels of the thyroid hormones
18    (triiodothyronine, T3, and thyroxine, T4)  and thyroid stimulating hormone (TSH) (or thyrotropin)
19    produced by the pituitary, and neurobehavioral outcomes measured using validated instruments in
20    infants and children.
21          The details of the laboratory procedures, including information on the basic methods, limit
22    of detection, and coefficient of variation, are important considerations for the hormone assays.
23    Thyroid hormones are generally measured in serum, although they may also be measured in whole
24    dried blood spots, such as are collected from newborn infants in screening for congenital
25    hypothyroidism as well as for genetic metabolic diseases  such as phenylketonuria. Studies in older
26    age groups have also shown a high correlation between thyroid hormone levels measured in dried
27    blood spots and levels in serum (Hofman  et al., 2003].
28          With respect to thyroid hormones, time of day and season are two potential sources of
29    variability. For example, serum TSH measured shortly after midnight may be as much as twice as
30    high as the value measured in late afternoon (Brabant etal., 1991: Weeke and Gundersen,  1978].
31    The evidence with respect to seasonal variability is mixed (Plasquietal.. 2003: Maes etal.. 1997:
32    Nicolau etal.. 1992: Simoni etal.. 1990: Behall etal.. 1984: Postmes etal.. 1974] and this effect is
33    likely to be smaller than that of time of day.  The impactofthese sources of variation will depend on
34    whether they are also related to  HBCD (i.e., do HBCD levels vary by time of day or season?]. If this
35    is the case, failure to address these factors in the design or analysis could result in confounding of
36    the observed association, with the direction determined by the direction of the association between
37    these factors and HBCD.  If this is not the case, the lack of consideration of time of day or seasonality
38    would result in greater variability in the hormone measures, and thus would result in more
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    imprecise (but not biased) estimates. EPA has not found evidence of a seasonal or diurnal variation
 2    in HBCD levels, and thus considers the latter scenario, i.e., lack of consideration of these factors
 3    leading to greater imprecision, rather than a biased effect estimate, to be more likely.
 4           With respect to neurodevelopmental outcomes, a major consideration is the assessment
 5    tool(s) used by the study investigators; details of the assessment method, or references providing
 6    this information, should be provided. In addition, EPA also looks for discussion of (or reference to)
 7    validation studies and the appropriateness of the tool for evaluation in the specific study population
 8    (e.g., age range, language).
 9
10    Confounding
11           The general considerations for evaluating issues relating to potential confounding include
12    consideration of which factors may be potential confounders (i.e., those that are strongly related to
13    both the exposure and the outcome under consideration), and if needed, control for these potential
14    confounders in the study design or analysis.  Adequacy of the measurement of confounders, and the
15    potential for residual confounding, will also be considered.
16           Age and sex are considered important explanatory factors for the hormone measures, as
17    well as for the neuropsychological and neurobehavioral outcomes, even in the absence of strong
18    associations with HBCD (Rawnetal., 2014).  A measure of socioeconomic status (e.g., parental
19    education level) is also typically used in studies  of cognition and behavioral outcomes, although
20    associations between HBCD and socioeconomic  status have not been established.
21
22    Analysis
23           The general considerations for evaluating issues relating to analysis are outlined in the draft
24    Preamble.  These include adequate documentation of analytic approach to interpret study results,
25    consideration of sample size and statistical power, and use of appropriate methods for the study
26    design.
27           As noted above, a major analytic consideration is how lack of variability in the exposure
28    and/or the outcome(s) is addressed—for example, as may occur if many HBCD measurements fall
29    below the LOD. The study should describe the distribution of HBCD exposure andoutcome(s) in
30    the study population (for both the study and comparison groups), and if needed, use appropriate
31    analytic techniques to address lack of variability, or unusual or skewed distributions.
32
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
           Table 3-1. General and outcome-specific considerations for HBCD human
           study evaluation
General considerations
Study population
Exposure
Analysis
- Study population and setting: geographic area, site, time period, age and sex
distribution, other details as needed (may include race/ethnicity, socioeconomic
status)
- Recruitment process; exclusion and inclusion criteria, knowledge of study
hypothesis, knowledge of exposure and outcome
- Participation rates: Total eligible, participation at each stage and for final analysis
group and denominators used to make these calculations
Length of follow-up, loss to follow-up
- Comparability: Participant characteristic data by group, data on non-participants
- Specific HBCD isomer(s) measured
- Limit of detection (LOD) or level of quantitation (LOQ)
Exposure distribution (e.g., central tendency, range), proportion < LOD
- Consideration of skewness of exposure and outcome measures
- Consideration of values below LOD or LOQ
- Consideration of lipids (for serum or breast milk samples) adjustment
Presentation of quantitative results, rather than statement regarding presence or
absence of statistical significance
Outcome-specific considerations
Neuropsychological
and neurobehavioral
Measures
Consideration of
confounding
Thyroid
Measures
Consideration of
confounding
- Standardized assessment tool, validation studies for specific study population (e.g.,
age group, geographic location)
Blinding of assessor to exposure
- Age, sex, socioeconomic status
- Assay used and evidence from validation studies, if available
- Sensitivity/detection limits, coefficient of variation; number of samples below LOD
- Biological sample used (e.g., serum, dried whole blood spots)
- Time of day and season when samples for thyroid hormone (and TSH) collected
- Age, sex
1
2
3
4
5
6
7
3.4.2.  Experimental Animal Studies
       Beyond the initial methodological screening described above in Section 3.2.2,
methodological aspects of a study's design and conduct will be considered again in the overall
evaluation and synthesis of the pertinent data that will be developed for each health effect Some
general questions that will be considered in evaluating experimental animal studies are presented
in Table 3-2. These questions are, for the most part, broadly applicable to all experimental studies.
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                                     Preliminary Materials for the IRIS Toxicological Review ofHBCD
             Table 3-2. Questions and relevant experimental information for evaluation of
             experimental animal studies
        Methodological
           featu re
                              Question(s) considered
          Examples of relevant
         information extracted
     Test animal
                     Based on the endpoint(s) in question, are
                     concerns raised regarding the suitability of
                     the species, strain, or sex of the test
                     animals on study?
Test animal species, strain, sex
     Experimental setup
                     Are the timing, frequency and duration of
                     exposure, as well as animal age and
                     experimental group allocation procedures/
                     group size for each endpoint evaluation,
                     appropriate for the assessed endpoint(s)?
Age/lifestage of test animals at exposure
and all endpoint testing timepoints

Timing and periodicity of exposure and
endpoint evaluations; duration of exposure

Experimental group allocation procedures
and sample size for each experimental
group (e.g., animals; litters; dams) at each
endpoint evaluation
     Exposure
                     Are the exposure conditions and controls
                     informative and reliable for the endpoint(s)
                     in question, and are they sufficiently
                     specific to the compound of interest?
Test article composition, stability, and
vehicle control

Exposure administration techniques (e.g.,
route; chamber type) and related controls
     Endpoint evaluation
     procedures
                     Do the procedures used to evaluate the
                     endpoint(s) in question conform to
                     established protocols, or are they
                     biologically sound? Are they sensitive for
                     examination of the outcome(s) of interest?
Specific methods for assessing the effect(s)
of exposure, including related details (e.g.,
biological matrix or specific region of
tissue/organ evaluated)

Endpoint evaluation controls, including
those put in place to minimize evaluator
bias
     Outcomes and data
     reporting
                     Were data reported for all pre-specified
                     endpoint(s) and study groups, or were any
                     data excluded from presentation/
                     analyses?
Data presentation for endpoint(s) of
interest
1
2
3
4
5
6
7
8
Note: "Outcome" refers to findings from an evaluation (e.g., steatosis), whereas "endpoint" refers to the
evaluation itself (e.g., liver histopathology).

        Evaluation of some specific methodological features identified in Table 3-2, such as
exposure, is likely to be relatively independent of outcome. Other methodological features, in
particular those related to experimental setup and endpoint evaluation procedures, are generally
outcome specific (e.g., reproductive and developmental toxicity). Some specific aspects of study
methodology that will be considered in the evaluation and synthesis of the HBCD literature are
described below.
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1    Exposure
 2           Commercial HBCD consists of three primary isomers (a, (3, and y). Because these isomers
 3    display different toxicokinetic properties, the isomeric composition of the test material could
 4    influence study results. Accordingly, the isomeric composition tested in each study will be
 5    considered in the development of the synthesis. Information on purity of the commercial mixtures
 6    may be important as well. Information on the test material as reported by study authors for those
 7    experimental animal studies included in preliminary evidence tables is summarized in Appendix B
 8    (Table A-8).
 9           The majority of studies administered HBCD in the diet  Because HBCD is semivolatile and
10    can partition into the atmosphere when exposed to air, documentation of stability of the test
11    material in the diet will be a consideration.
12
13    Outcome-specific Considerations
14           In general, experimental animal studies will be compared against traditional assay formats
15    (e.g., those used in guideline studies), with deviations from the protocol evaluated in light of how
16    the deviations could alter interpretation of the outcome in question. A number of the HBCD studies
17    applied study protocols to examine effects of HBCD on the thyroid, nervous system, reproduction,
18    development, and immune system.
19
20    Thyroid Endpoints
21           The HBCD experimental animal database includes several studies of the  potential effects of
22    HBCD on the thyroid, and in particular thyroid hormone level testing. Specific Agency guidelines on
23    testing and evaluation of thyroid endpoints are not available. Some considerations for evaluating
24    studies of thyroid endpoints include the following:
25       •   Radioimmunoassays (RIA) are generally the standard for measuring thyroid hormones in
26           rodent studies. Results from ELIZA assays should be interpreted cautiously; reported
27           detection limits should be based on within-laboratory calibrations and not on assay specs.
28           [The specific assay used in the studies that measured thyroid hormones  are reported in the
29           preliminary evidence tables.]
30       •   Hormones should be sampled at the same time of day because of fluctuations in T3 and T4
31           levels throughout the day in rats.
32       •   Whether both male and female animals were tested because of possible  gender differences
33           (e.g., differences  associated with maturation of reproductive hormone systems and cyclicity
34           in females).
35       •   Hormone testing protocols will be evaluated further for other experimental setup features
36           and endpoint evaluation procedures, including sensitivity/detection limit calibrations for
37           each assay, validation of assays using heterologous antibodies, extent of outcomes below
38           the limit of detection (LOD), and minimization of nontreatment-related influences on

           This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1           hormone levels (e.g., stress-induced alterations, thyroid hormone suppression from
 2           anesthesia), and inclusion of positive control treatment with expected serum thyroid
 3           hormone pattern (e.g., methimazole or propylthiouracil).
 4
 5    Neurological and Neurobehavioral Endpoints
 6           The HBCD experimental database includes functional observational batteries (FOB) in adult
 7    rats, and protocols to examine motor function-related behaviors and cognition (memory assessed
 8    using water maze) in rat pups in a multigenerational study, locomotor activity in mice pups, and
 9    electrophysiology in rats following developmental exposure. In general, assays used in studies will
10    be compared to traditional assay formats for evaluating these specific neurotoxicity endpoints.
11           EPA's Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998a) outline important
12    aspects of study design that should be considered when assessing neurotoxicity endpoints; as
13    applicable, these guidelines will be used for characterizing and interpreting assay results from
14    neurotoxicity studies. For those studies of neurotoxicity endpoints evaluated in experimental
15    animals exposed during development, additional considerations provided in EPA's Guidelines for
16    Developmental Toxicity Risk Assessment (U.S. EPA, 1991) are applicable. In addition, the following
17    considerations regarding specific tests employed in the HBCD database will be incorporated:
18       •   For all behavioral assays, it is desirable for the investigators recording the responses to be
19           blinded as to the treatment of the test animals.
20       •   Tests of motor activity should be of sufficient duration (e.g., >20 minutes), and should be
21           evaluated in the absence of evidence of systemic toxicity, as this may cause
22           misinterpretation due to nonneurotoxic effects. While tests of shorter duration may still be
23           useful, consideration should be paid to the involvement of behaviors other than motor
24           function. For non-developmental evaluations of motor function (e.g., coordination and
25           dexterity), it is desirable that the results be presented as continuous rather than (or in
26           addition to) dichotomized data, as it is problematic to arbitrarily define a response as a
27           "success" or "failure," particularly without first establishing a baseline across a large
28           number of animals with the same phenotype, housing, and test conditions.
29       •   FOBs typically represent a standardized series of tests evaluating various domains of
30           nervous system function within a short time period (e.g., 10 minutes). While most studies
31           use a validated FOB design, application of the FOB can vary across laboratories, which can
32           introduce additional uncertainty. For example, an FOB should take care to consider and
33           account for the order of testing, as order effects in these batteries can introduce nonspecific
34           effects.
35       •   Measurements of memory and learning should be separated from other changes in behavior
36           that do not involve cognitive processes (e.g., motor function).  Specifically regarding water
37           maze tests, the temperature of the water bath, platform and pool size, and visual cues
38           (including the investigator) necessary for  accomplishing the task should be controlled for

           This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1          and appropriate to the test animal species and age under investigation.
 2
 3    Reproductive and developmental endpoints
 4          The HBCD database includes 1- and 2-generation reproductive toxicity studies, a
 5    developmental study, and other repeat-dose studies that examined reproductive organs. EPA's
 6    Guidelines for Reproductive Toxicity Risk Assessment [U.S. EPA. 1996] detail study design
 7    parameters that are of particular importance in reproductive toxicity studies. These factors include
 8    duration of dosing, length of mating period and number of males and females mated; type of test
 9    (single versus multigeneration studies); and endpoints evaluated.  Test guidelines for the conduct
10    of single- and multigene ration reproduction protocols that have been published by EPA and OECD
11    will be utilized in evaluation of the reproductive and developmental toxicity database for HBCD
12    fU.S. EPA. 1996. 1985: Galbraith etal.. 1983: OECD.  19831
13          Likewise, EPA's Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA. 19911
14    detail study design parameters that are of particular importance in developmental toxicity studies.
15    Evaluation of developmental endpoints includes studies that typically involve exposure of pregnant
16    animals during critical windows of organo gene sis, evaluation of maternal toxicity throughout
17    pregnancy, and examination of dams and uterine contents [U.S. EPA, 1991]. Developmental toxicity
18    studies also may evaluate exposures of one to a few days to investigate critical windows of
19    development  Endpoints typically evaluated in developmental toxicity studies include assessment
20    of maternal toxicity, altered survival and growth, morphological development, and functional
21    deficits. A particular consideration in  developmental toxicity studies is the selection of a high dose
22    that produces minimal maternal or adult toxicity (i.e., a level that at the least produces marginal but
23    significantly reduced body weight, reduced weight gain, or specific organ toxicity, and at the most
24    produces no more than 10% mortality]. At doses that cause excessive maternal toxicity (that is,
25    significantly greater than the minimal toxic level], information on developmental effects may be
26    difficult to interpret and of limited value.
27
28    Immune endpoints
29          The HBCD database includes limited testing of immunotoxic potential, largely focused on
30    cell counting, and functional immune assays.  In general, functional assays will be weighed more
31    heavily than observational endpoints such as cell counts and organ weights.  Immuno toxicity
32    testing guidelines will be used to evaluate adherence to established protocols and to incorporate
33    current guidance practices for assessing immune endpoings, including the following:
34       •   WHO/International Programme on Chemical Safety (IPCS] Harmonization Project
35          Document No. 10, Guidance for Immunotoxicity Risk Assessment for Chemicals (WHO.
36          2012]  (available at
37          http://www.inchem.org/documents/harmproj/harmproj/harmprojlO.pdf].
38       •   WHO/International Programme on Chemical Safety (IPCS] Environmental Health Criteria
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1           180: Principles and Methods for Assessing Direct Immunotoxicity Associated with Exposure
 2           to Chemicals (WHO, 1996) (available at
 3           http://www.inchem.org/documents/ehc/ehc/ehcl80.htm].
 4       •   U.S. EPA Health Effects Test Guidelines, OPPTS 870.7800, Immunotoxicity (U.S. EPA. 1998b)
 5           (available athttp://www.regulations.gov/#!documentDetail:D=EPA-HO-OPPT-2009-0156-
 6           00491.
 7       •   OECD Test Guidelines 443 (Extended One-Generation Reproductive Toxicity Study test
 8           guideline), 407 (Repeated Dose 28-day Oral Toxicity Study in Rodents test guideline), 408
 9           (Repeat Dose 90-day Oral Toxicity in Rodents test guideline), and 413 (Subchronic
10           Inhalation Toxicity: 90- Day Study test guideline), which include endpoints that may give an
11           indication of immunological effects or, in the case of Test Guideline 443, developmental
12           immunotoxicity. (available at
13           http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm).

14           A full evaluation of all pertinent studies will be performed as part of the critical review and
15    synthesis of evidence for hazard identification for each of the health endpoints identified in the
16    evidence tables (Appendix A).
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                                     Preliminary Materials for the IRIS Toxicological Review ofHBCD
      4.   REFERENCE  LIST
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17    Ethyl Corporation. (1990). Genetic toxicology rat hepatocyte primary culture/DNA repair test on
18            hexabromocyclododecane with cover letter dated 030890. Baton Rouge, LA.
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23    Fernandez Canton. R: Sanderson. T: Nijmeijer. S: Bergman. A: Van Den Berg. M. (2005). In vitro effects of
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27            the human conceptus from exposure to environmental substances. In Advances in Modern
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29    Geyer. HI: Schramm. K. -W: Darnerud. PO: Aune. M: Feicht. EA: Fried. KW: Henkelmann. B: Lenoir. D: Schmid.
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32    Harju. M: Hamers. T: Kamstra. IH: Sonneveld. E: Boon. IP: Tysklind. M: Andersson. PL. (2007). Quantitative
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35            http://dx.doi.Org/10.1897/06-308R.l

36    Helleday. T: Tuominen. KL: Bergman. A: lenssen. D. (1999). Brominated flame retardants induce intragenic
37            recombination in mammalian cells. Mutat Res 439:137-147.

38    Hinkson. NC: Whalen. MM. (2009). Hexabromocyclododecane decreases the lytic function and ATP levels of
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40    Hinkson. NC: Whalen. MM. (2010). Hexabromocyclododecane decreases tumor-cell-binding capacity and cell-
41            surface protein expression of human natural killer cells. J Appl Toxicol 30: 302-309.
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43    Hoftnan. LF: Foley. TP: Henry. II: Naylor. EW. (2003). Assays for thyroid-stimulating hormone using dried
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            This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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 1    Hooper. K: She. I: Sharp. M: Chow. I: lewell. N: Gephart. R: Holden. A. (2007). Depuration of polybrominated
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39    Koike. E: Yanagisawa.  R: Takigami. H: Takano. H. (2012). Brominated flame retardants stimulate mouse
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25    Wu. M: Zuo. Z: Li.  B: Huang. L: Chen. M: Wang. C. (2013). Effects of low-level hexabromocyclododecane
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD


1    Zhang. X: Yang. F: Zhang. X: Xu. Y: Liao. T: Song. S: Wang. I. (2008b). Induction of hepatic enzymes and
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5

6
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                               Preliminary Materials for the IRIS Toxicological Review ofHBCD

 1
     APPENDIX A.   PRELIMINARY EVIDENCE TABLES
     AND EXPOSURE-RESPONSE ARRAYS
 2   A.I. Data Extraction: Preparation of Preliminary Evidence Tables and
 3        Exposure-Response Arrays for Primary Studies
 4         Key study design information, including study characteristics that inform the quality of the
 5   studies, and results from primary sources of health effects data considered pertinent for evaluating
 6   the health effects from chronic exposure to HBCD are summarized in preliminary evidence tables
 7   (Appendix A). The information in the preliminary evidence tables is also displayed graphically in
 8   preliminary exposure-response arrays.  In these arrays, a significant effect (indicated by a filled
 9   circle) is based on statistical significance.
10         Key study design information and results from human studies are summarized in a single
11   preliminary evidence table (Table A-l) rather than in multiple tables by health effect because the
12   outcomes examined in these studies, including endocrine (thyroid and reproductive hormone),
13   neuropsychological, neurobehavioral, and developmental outcomes may be inter-related.
14   Considering the human studies as a group may provide a more integrated evaluation of the
15   potential health effects of HBCD. In addition, human evidence will be considered together with the
16   available animal evidence in the overall evaluation and synthesis of evidence for each health effect.
17         The complete list of references considered in preparation of these materials can be found on
18   the HERO website at http://hero.epa.gov/HBCD.
19
20
          This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                      Preliminary Materials for the IRIS Toxicological Review ofHBCD


1    A.2.  Effects in Humans

            Table A-l.  Evidence pertaining to effects in humans
               Reference and Study Design
                        Results
     Studies in infants and children
     Eggesb0 et al. (2011) (Norway, 2003-2006)
Association between HBCD level in breast milk with neonatal
TSH levels:
     Birth cohort, recruited within 2 weeks of delivery
     study (able and willing to provide breast milk
     sample), 396 randomly selected for analysis; 234 of
     these were after Feb 2004 when link to thyroid data
     became available; 193 with HBCD data (46% girls)

     Exposure measures: breast milk, collected at a
     median of 33 days after delivery (samples pooled
     over 8 consecutive mornings)
     HBCD detected in 67.9% of samples
     LOQ = 0.2 ng/g lipid
     Median 0.54 (range: 0.1-31) ng/g lipid

     Effect measures: TSH (whole blood spots) measured
     in infants 3 days after delivery; immunoassay
     (clinical lab)

     Analysis: Linear regression with In TSH as a
     continuous outcome and logistic regression with
     dichotomized In TSH (at 80thpercentile); see results
     column for consideration of covariates. Referent
     category includes all samples < LOQ; remaining 32%
     of population divided into 4 other categories.
                         Adjusted Beta
                           (95% Cl)a
                  Adjusted odds
                  ratio (95% Cl)b
  0.1 ng/g lipid
  0.13-0.52 ng/g lipid
  0.53-0.79 ng/g lipid
  0.80-1.24 ng/g lipid

  1.29-31.2 ng/g lipid
  Per IQR increase:
    (Referent)

-0.01 (-0.21, 0.20)

0.02 (-0.18, 0.22)

0.12 (-0.08, 0.33)

0.03 (-0.17, 0.23)

-0.00 (-0.02, 0.02)
 (Referent)

1.3 (0.3, 4.5)

1.4(0.3,6.1)

1.6 (0.4, 6.1)

1.3 (0.3, 5.8)

1.0 (0.8, 1.1)
  a outcome = In TSH; 0.0 = no effect
  b outcome = TSH >80th percentile; 1.0 = no effect
  Adjusted for age at TSH screening, maternal BMI, county,
  ppDDE, HCB, delivery type, pregnancy preeclampsia and
  hypertension. Also evaluated but eliminated maternal
  education, age at delivery, Norwegian nationality, season,
  parity, smoking, sex, gestational age, beta-HCH,
  oxychlordane, and sum of all PCB congeners.
     Meijer et al. (2012) (the Netherlands, COMPARE
     cohort, 2001-2002)

     Pregnancy cohort, 90 singleton, term births, 55
     healthy boys, assessed at 3 months (n=55) and 18
     months (n=52); 44 with HBCD measures, 45 with
     hormone measures, 34 with both measures

     Exposure measures: prenatal exposure, maternal
     serum at 35th week of pregnancy
     1,2,5,6,9,10-HBCD (HBCDD) detected in 43 of 44
     samples
     LOD 0.8 pg/g serum; LOQ = 9 pg/g serum
     Median 76 (range 36-180) pg/g serum or 0.7 (range:
     n.d.-7.4) ng/g lipid

     Effect measures:
     Hormones (serum, collected at 3 months)
     (immunoassay details in  Laven et al., 2004)
Spearman correlation between HBCDD in maternal serum and
free testosterone: r = -0.31 (0.05 < p-value < 0.10)

Correlations with other hormones noted as not statistically
significant but quantitative results were not reported

No significant correlations between prenatal exposure to
HBCD and testes volume or penile length were found (data
not shown).
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
        Table A-l. Evidence pertaining to effects in humans
           Reference and Study Design
                        Results
 • testosterone
 • sex hormone binding globulin (SHBG)
 • follicle stimulating hormone (FSH)
 • luteinizing hormone (LH)
 • estradiol (E2)
 • inhibin B
 Testes volume, measured by ultrasound (ages 3
 and 18 months); penile length (ages 3 and 18
 months)

Analysis: Spearman correlation
Roze et al. (2009) (the Netherlands, COMPARE
cohort, 2001-2002 at baseline)

Pregnancy cohort, 90 singleton, term births, 62 of
69 (90%) mother-child pairs randomly selected  from
the cohort for HBCDD measures in serum; children
ages 5-6 years at follow-up

Exposure measures: Prenatal exposure, maternal
serum at 35th week of pregnancy
1,2,5,6,9,10-HBCD (HBCDD) detected in all samples
LOD 0.8 pg/g serum; LOQ = 9 pg/g serum
Median 0.8 (range: 0.3-7.5) ng/g lipids

Effect measures:
Neuropsychological tests (references for procedure
provided)
 • Movement ABC test battery for motor
   performance (coordination, fine motor skills)
 • Developmental Coordination Disorder
   Questionnaire for behavior
 • Wechsler Preschool and Primary Scale of
   Intelligence, Revised for intelligence (total,
   verbal, performance)
 • Neuropsychological Assessment (NEPSY-II) for
   visual perception, visuomotor integration,
   inhibitory control
 • Rey's Auditory Verbal Learning test (verbal
   memory)
 • Test of Everyday Attention for Children
   (attention)
Behavioral tests (references for procedure
provided)
 • Child Behavior Checklist and Teacher's Report
   Form
 • Attention Deficit/Hyperactivity Disorder
Neuropsychological measure   Correlation coefficient"
   Coordination                   0.29 (p<0.05)
   Total intelligence              0.393  (p<0.05)
   Verbal intelligence             0.479  (p<0.01)
 a positive correlations indicate better outcomes.
 Correlations between lipid-adjusted HBCDD and outcome
 measure adjusted for SES, Home Observation for
 Measurement of the Environment HOME score, and sex.

Results for correlations between other outcomes
(neuropsychological, behavioral and thyroid hormone levels)
were not shown, but were stated to be not statistically
significant (p>0.10).
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
        Table A-l.  Evidence pertaining to effects in humans
           Reference and Study Design
                        Results
   questionnaire
Hormones (cord blood samples, n=51, selected
based on amount of sample available):
  T4, freeT4, rT3, T3, TSH, TBG (assay not described)

Analysis: Pearson correlation (for normally
distributed variables) or Spearman's rank
correlation (for non-normally distributed variables)
Studies in adolescents
Kicihskietal. (2012) (Belgium, 2008-2011)
Cross-sectional study, 515 adolescents (13-17 yr
old) from two industrial sites and randomly selected
from the general population; participation rates 22-
34% in the 3 groups, sample size varies by test

Exposure measures: Serum samples, HBCD
> 75% were < LOQ (LOQ = 30 ng/L);
Median <30 (range:  LOQ versus  LOQ versus 
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
        Table A-l.  Evidence pertaining to effects in humans
          Reference and Study Design
                        Results
                                               home ownership, physical activity, computer use, alcohol
                                               and fish consumption, blood lead and blood PCBs, and were
                                               included based on a stepwise regression procedure.
Studies in adult men
Johnson etal. (2013) (USA, 2002-2003)
Cross-sectional study, 38 men (18-54 yr old), from
couples seeking infertility treatment; approximately
65% participation into general study; participation
rate in the vacuum bag collection phase not
reported

Exposure measures: HBCD exposure from vacuum
bag dust; three main stereoisomers of HBCD
presented together.
HBCD detected in 97% of samples;  LOD not
reported;
median 246 (90th percentile 1103) ng/g dust

Effect measures:
Non-fasting blood sample (immunoassay details in
Meeker et al., 2008)
 • testosterone (T)
 • sex hormone binding globulin (SHBG)
 • follicle stimulating hormone (FSH)
 • luteinizing hormone (LH)
 • estradiol (E2)
 • inhibin B
 • prolactin
 • free T4
 • free T3
 • thyrotropin (TSH)

Analysis:  All variables analyzed as continuous
variables; Spearman's correlation between HBCD in
house dust and serum hormone  levels;
multivariable models adjusted for age  and BMI
Free androgen index
(T/SHBG)
Correlation coefficient"

0.46 (p=0.004)
SHBG                 -0.35a(p=0.03)
a negative values indicate inverse correlations

Results for other hormones not shown. Results from
regression models not shown, but reported to be similar to
bivariate correlations.

Note that HBCD was not strongly correlated with other flame
retardants measured (Spearman correlation coefficients
ranging from-0.20 to 0.27, all p-values >0.10)
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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
1    A.3. Effects in Animals
2          The evidence tables present data from studies related to a specific outcome or endpoint of
3    toxicity.  Information in the preliminary evidence tables is also displayed graphically in preliminary
4    exposure-response arrays.  In these arrays, a significant effect is based on statistical significance,
5    with significantly different effects at individual doses based on a pairwise comparison indicated by
6    a filled circle, or significant dose-related trends indicated by filled circles at all doses.

7    A.3.1. Thyroid Effects Evidence Table and  Exposure-response Array
8          Table A-2. Evidence pertaining to thyroid effects in animals following oral
9          exposure to HBCD
Reference and Study Design
Results
Thyroid hormones
(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGS BR rats, 20-40/sex/group
0, 100, 300, 1,000 mg/kg-d
Gavage
90 d (13 wks) with additional 28-d (4-wk)
recovery period
Method used to measure thyroid hormones was
not reported.
van derVen et al. (2006)
Wistar rats, 5/sex/group (3-5/sex/group for
thyroid hormones)
0, 0.3, 1, 3, 10, 30, 100, 200 mg/kg-d
Gavage
Percent change compared to control"
Doses 0
100
300
1000
T3 (wk 13)
M
F
0%
0%
-9%
-4%
-8%
-9%
0%
-4%
T3 Recovery (wk 17)
M
F
0%
0%
22%
-1%
11%
6%
28%
17%
T4 (wk 13)
M
F
0%
0%
-19*%
-9%
-20*%
-17*%
-37*%
-21*%
T4 Recovery (wk 17)
M
F
0%
0%
2%
14%
10%
14%
-14%
25%
TSH (wk 13)
M
F
0%
0%
1043*%
396*%
1052*%
1448*%
1587*%
957*%
TSH Recovery (wk 17)
M
F
0%
0%
-75%
-3%
-57%
-32%
-15%
24%
Percent change compared to control"
Doses 0 0.3
1 3
10 30
100 200
TT3
M 0% 4%
F 0% -8%
5% 10%
-3% -11%
20% 11%
-12% -19%
1% 10%
1% -10%
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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design
28 d
Thyroid hormones measured by
radioimmunoassay
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and F2
offspring produced, serum hormone levels were
measured in FO and Fl adults only (8/sex/group)
0, 150, 1,500, 15,000 ppm (mean daily intakes):
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
Diet
10 wks prior to mating and through gestation,
lactation, and for two generations (multi-
generation reproductive toxicity study)
Thyroid hormones measured by
radioimmunoassay
Saegusa et al. (2009)
Crj:CD(SD)IGS rats, 10 dams/group, litters culled
to 4/sex/dam on PND 2, Fl animals maintained
for 11 wks, only male offspring evaluated for
thyroid hormones
0, 100, 1,000, 10,000 ppm (TWAC: 0, 14.8, 146.3,
1,505 mg/kg-d)
Diet
GD10-PND20
Thyroid hormones measured by
electrochemiluminescence immunoassay
Results
TT4
M 0% 1% 1% 23%
Fb 0% 2% -3% -10%
8% 5%
-7% -8%
-13% 3%
-13% 26**%
Percent change compared to control"
Doses Oppm ISOppm
ISOOppm
ISOOOppm
T3 FO Adults
M 0% -4%
F 0% 6%
-15%
10%
-12%
2%
T3 Fl Adults
M 0% 1%
F 0% -2%
2%
-10%
0%
-11%
T4 FO Adults
M 0% -2%
F 0% 11%
-27%
6%
-38*%
-31*%
T4 Fl Adults
M 0% -3%
F 0% -1%
-6%
-6%
-10%
-28%
TSHFO Adults
M 0% 0%
F 0% 39*%
19%
44*%
44%
102*%
TSH Fl Adults
M 0% -4%
F 0% 48%
32%
75%
30%
67*%
Percent change compared to control"
Doses 0 14.8
146.3
1505
T3F1 weanling (PND 20)
M 0% 4%
-3%
-15*%
T3 Fl adults (wk 11)
M 0% -3%
-8*%
-7*%
T4F1 weanling (PND 20)
M 0% -4%
9%
-4%
T4 Fl adults (wk 11)
M 0% 2%
9%
9%
TSH Fl weanling (PND 20)
M 0% 23%
12%
30*%
TSH Fl adults (wk 11)
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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design

Results
M 0% 23% 13% 5%
Thyroid weight
(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGS BR rats, 10/sex/group
0, 100, 300, 1,000 mg/kg-d
Gavage
90 d (13 wks) with additional 28-d (4-wk)
recovery period
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and F2
offspring produced, thyroid weight was
measured in FO and Fl adults only (13—
24/sex/group)
0, 150, 1,500, 15,000 ppm (mean daily intakes):
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
Diet
10 wks prior to mating and through gestation,
lactation, and for two generations (multi-
generation reproductive toxicity study)
Saegusa et al. (2009)
Crj:CD(SD)IGS rats, 10 dams/group; litters culled
to 4/sex/dam on PND 2, Fl animals maintained
for 11 wks (10/sex/group for thyroid weight in
FOand Fl adults)
0, 100, 1,000, 10,000 ppm (TWAC: 0, 14.8, 146.3,
1,505 mg/kg-d)
Diet
GD10-PND20
Percent change compared to control"
Doses 0 100 300 1000
Absolute thyroid weight (wk 13)
M
F
0% 20% 12% 0%
0% 14% 6% 15%
Thyroid/body weight (wk 13)
M
F
0% 0% 0% 0%
0% 17% 0% 17%
Absolute thyroid weight (wk 17)
M
F
0% 7% -2% -3%
0% 25% 36*% 37*%
Thyroid/body weight (wk 17)
M
F
0% 20% 0% 20%
0% 17% 33*% 33*%
Percent change compared to control"
Doses 0 ppm 150 ppm 1500 ppm 15000 ppm
FO adults
M
significant increases in absolute and relative
thyroid weight
Absolute thyroid weight Fl adults
M
F
0% 0% 5% 19*%
0% 3% 11% 24*%
Thyroid/body weight Fl adults
M
F
0% 5% 3% 23*%
0% 1% 9% 29*%
Percent change compared to control"
Doses 0 14.8 146.3 1505
Thyroid/body weight FO Adults
M
F
0% 18% 10% 30*%
Thyroid/body weight Fl Adults
M
F
0% 17% 19*% 28*%
0% -17% -10% -6
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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design
Saegusa et al. (2012)
Crj:CD(SD)IGS rats, 10 dams/group
Fl: 20/sex/group
0, 100, 1,000, 10,000 ppm (0, 14.8, 146.3, 1,505
mg/kg-d)d; 1,2,5,6,9, 10-H BCD
Diet
GD10-PND20
Thyroid histopathology
(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGS BR rats, 20-40/sex/group
0, 100, 300, 1,000 mg/kg-d
Gavage
90 d
Maranghi et al. (2013)
BALB/c female mice
0 (15/group), 199 mg/kg-d (10/group)
Diet
28 d
BASF (1990)
Sprague-Dawley rats, 10/sex/group
(5/sex/group for thyroid histopathology)
0, 1, 2.5, 5.0% (males: 0, 900, 2,400,
4,700 mg/kg-d; females: 0, 900, 2,300,
4,900 mg/kg-d )c
Diet
28 d
(WIL Research Labs (1998), 1997))
Crl:CD(SD)BR rats (6-12/sex/group)
0, 125, 350, 1,000 mg/kg-d
Gavage
28 d




Results
Fl Results:
PND 20: statistically significant increased relative thyroid weight
at 1,505 mg/kg-d (data not provided)
PND 77: statistically significant increased relative thyroid weight
at 146.3 and 1,505 mg/kg-d (data not provided)

Incidence
Doses 0 100 300 1000
Thyroid follicularcell hypertrophy6
M 1/10 1/10 5/10 8/9**
F 0/10 0/10 4/10 7/10**
Doses 0 199
Ratio (follicle:colloid) 0 9*%
Follicle area 0% -20%
Colloid area 0% -26%
Reported dose-related increase in thyroid hyperplasia, with
highest dose characterized as having "very marked hyperplastic
thyroid tissue"; adenomatous proliferation and epithelial
hyperactivity in high-dose group; incidence data were not
provided.
Incidence
Doses 0 125 350 1000
Thyroid follicularcell hypertrophy6
Minimal
M 6/6 3/6 4/6 6/6
F 6/6 5/6 6/6 6/6
Mild
M 0/6 3/6 2/6 0/6
F 0/6 0/6 0/6 0/6
Colloid loss3
Minimal
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design

Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and F2
offspring produced, thyroids examined in FO and
Fl adults and Fl and F2 rats at weaning
0, 150, 1,500, 15,000 ppm (mean daily intakes):
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
Diet
10 wks prior to mating and through gestation,
lactation, and for two generations (multi-
generation reproductive toxicity study)
Saegusa et al. (2009)
Crj:CD(SD)IGS rats, 10 dams/group, litters culled
to 4/sex/dam on PND 2, Fl animals maintained
for 11 wks (10/sex/group for thyroid
histopathology)
0, 100, 1,000, 10,000 ppm (TWAC: 0, 15, 146,
1,505 mg/kg-d)
Diet
GD10-PND20
Saegusa et al. (2012)
Crj:CD(SD)IGS rats, 10 dams/group
FI: 20/sex/group
0, 100, 1,000, 10,000 ppm (0, 14.8, 146.3, 1,505
mg/kg-d)d
Diet
GD10-PND20
Results
M 5/6 3/6 5/6
F 4/6 4/6 5/6
1/6
5/6
Mild/Moderate
M 0/6 1/6 1/6
F 0/6 0/6 1/6
5/6*
1/6
Incidence
Doses Oppm ISOppm ISOOppm
ISOOOppm
Decreased thyroid follicle size FO adults
M 0/24 0/24 6/24*
F 0/24 0/24 5/25*
20/23*
11/23*
Decreased thyroid follicle size Fl adults
M 0/24 0/24 2/22
F 0/24 1/24 5/24
11/24*
13/24*
Thyroid follicularcell hypertrophy FO adults
M 0/24 0/24 3/24
F 0/24 0/24 2/24
1/24
0/24
Thyroid follicularcell hypertrophy Fl adults
M 0/24 0/24 0/24
F 0/24 0/24 0/24
No treatment-related histopathological changes
Flor F2 weanlings.
0/24
0/24
in thyroids in
Incidence
Doses 0 15 146
1505
Thyroid follicularcell hypertrophy FO adults
M ...
F 3/10 5/10 6/10
9/10
No treatment-related histopathological changes were reported
in thyroids from exposed Fl rats.
Fl Results:
PND 20: increased follicular hypertrophy at 1,505
(statistically significant)
mg/kg-d
* Statistically significantly different from the control at p < 0.05, ** indicates p<0.01
a Percent change compared to control calculated as: (treated value - control value)/control value x 100.
 Significant dose response as reported by authors.
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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD
          Reference and Study Design                                     Results
CTWA doses were estimated based on food intake and body weight data (as reported by study authors).
d TWA doses calculated based on food intake and body weights measured in Saegusa et al. (2009).
e Pairwise significance tests were conducted by EPA. For incidence data, Fisher's Exact tests were used. All statistical analyses
 were conducted using the freely available R statistical software (version 3.0.1).  For continuous data (where means and
 standard deviations are provided), Student's T-tests were used.
GD = gestation day; PND = postnatal day; PNW = postnatal week; T3 = triidothyronine; T4 = thyroxine; TSH = thyroid stimulating
 hormone; TT3 = total triiodothyronine; TT4 = total thyroxine; TWA = time-weighted average
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                                                     Preliminary Materials for the IRIS Toxicological Review ofHBCD
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** Significant dose response as reported by study authors





        Figure A-l. Exposure-response array of thyroid effects following oral exposure to HBCD
                           This document is a preliminary draft for review purposes only and does not constitute Agency policy,

                                                              A-12                                 DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
1   A.3.2. Liver Effects Evidence Table and Exposure-response Array
2
3
Table A-3. Evidence pertaining to liver effects in animals following oral
exposure to HBCD
Reference and Study Design
Results
Liver histopathology
Kurakawa et al. (1984)
SLc;B6C3F! mice, 50/sex/group
0, 100, 1,000, 10,000 ppm (0, 17, 170,
1,720 mg/kg-d)a
Diet
18 mo
Pharmakologisches Inst (1990b)
Sprague-Dawley rats, 20/sex/group
0, 0.16, 0.32, 0.64, 1.28% (males: 0, 100,
200, 400, 900 mg/kg-d; females 0, 100,
200, 500, 950 mg/kg-d )c
Diet
13wks
(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGS BR rats, 15-35/sex/group
0, 100, 300, 1,000 mg/kg-d
Gavage
90 d
Maranghietal. (2013)
BALB/c female mice
0 (15/group), 199 mg/kg-d (10/group)
Diet
28 d
Incidence
Doses 0 17 170
1720
Liver nodules'3
M 14/50 23/50 32/50**
F 2/50 2/50 5/50
26/50*
6/50
Vacuolization and fatty changes'5
M 8/50 9/50 31/50**
F 17/50 19/50 20/50
20/50*
28/50*
Incidence
Doses (M) 0 100
Doses (F) 0 100
200 400
200 500
900
950
Liver fatty accumulation'3
M 4/20 8/20
F 10/20 11/20
11/20* 12/20*
9/20 19/20
19/20*
16/20
Disseminated adipose dropletsb
M 1/20 0/20
F 5/20 4/20
1/20 2/20
7/20 6/20
6/20
10/20
Incidence
Doses 0 100 300
Hepatocellular vacuolationb
M 2/10 6/
F 3/10 6/
Incidence
Doses
Vacuolation in hepatocytes
Pyknotic nuclei in hepatocytes
Periportal lymphocytic infiltration
Tissue congestion

10 5/10
10 5/10

0
0/10
0/10
0/10
0/10
1000

6/9
9/10*

199
5/8**
2/8
6/8**
6/8**
         This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                              A-13           DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design
Saegusa et al. (2009)
Crj:CD(SD)IGS rats, 10 dams/group;
litters culled to 4/sex/dam on PND 2, Fl
animals maintained for 11 wks
(10/sex/group for liver histopathology)
0, 100, 1,000, 10,000 ppm (TWAC 0, 14.8,
146.3, 1,505 mg/kg-d)
Diet
GD10-PND20
Liver weight
Pharmakologisches Inst (1990b)
Sprague-Dawley rats; 20/sex/group
0, 0.16, 0.32, 0.64, 1.28% (males: 0, 100,
200, 400, 900 mg/kg-d; females 0, 100,
200, 500, 950 mg/kg-d)
Diet
lo WKS

(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGS BR rats, 15-35/sex/group
(10/sex/group for liver weight)
, 100, 300, 1,000 mg/kg-d
Gavage
90 d (13 wks) with additional 28-d (4-wk)
recovery period





Results
Incidence
Doses 0 14.8
Hepatocellular vacuolar degeneration F1(PND
M 0/10 0/10
F 0/10 0/10


Percent change compared to control
Doses (M) 0 100 200
Doses (F) 0 100 200
Absolute liver weight b
M ...
F 0% 4**% 8**%
Liver/body weightb
M 0% 11**% 23**%
F 0% 5**% 10**%
Percent change compared to control
Doses 0 100
Absolute liver weight (wk 13)
M 0% 19*%
F 0% 22*%
Liver/body weight (wk 13)
M 0% 19*%
F 0% 24*%
Absolute liver weight (wk 17)
M 0% 2%
F 0% -6%
Liver/body weight (wk 17)
M 0% 12*%
F 0% -3%


146.3
20)
0/10
0/10



400
500

20**%

23**%
9**%

300

20*%
31*%

19*%
24*%

9%
9%

10*%
11%


1505

6/10*
6/10*



900
950

30**%

35**%
33**%

1000

33*%
53*%

44*%
48*%

-2%
13%

7%
12%
This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                     A-14           DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design
(WIL Research Labs (1998), 1997))
Crl:CD(SD)BR rats, 6-12/sex/group
(6/sex/group for liver weight)
0, 125, 350, 1,000 mg/kg-d
Gavage
28 d
BASF (1990)e
Sprague-Dawley rats, 5/sex/group for
liver weight
0, 1, 2.5, 5.0% (males: 0, 900, 2,400,
4,700 mg/kg-d; females: 0, 900, 2,300,
4,900 mg/kg-d )c
Diet
28 d
Maranghi et al. (2013)
BALB/c female mice
0 (15/group), 199 mg/kg-d (10/group)
Diet
28 d
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and
F2 offspring produced; liver weight was
assessed in all generations, 13-
24/sex/group
0, 150, 1,500, 15,000 ppm (mean daily
intakes):
FO males: 0, 10.2, 101, 1,008 mg/kg-d
FO females: 0, 14.0, 141, 1,363 mg/kg-d
Fl males: 0, 11.4, 115, 1,142 mg/kg-d
Fl females: 0, 14.3, 138, 1,363 mg/kg-d)
Diet
10 wks prior to mating and through
gestation, lactation, and for two
generations (multi-generation
reproductive toxicity study)
Results
Percent change compared to control
Doses 0 125
350
1000
Absolute liver weight
M 0% 6%
F 0% 18%
13%
29*%
25*%
40*%
Liver/body weight
M 0% 10%
F 0% 16*%
17*%
22*%
29*%
38*%
Percent change compared to control
Doses (M) 0 900
Doses (F) 0 900
2400
2300
4700
4900
Absolute liver weight
M 0% 39*%
F 0% 40*%
50*%
52*%
52*%
72*%
Liver/body weight
M 0% 27*%
F 0% 33*%
59*%
62*%
105*%
108*%
Percent change compared to control
Doses 0
Absolute liver weight 0%
Relative liver weight 0%
Doses Oppm ISOppm


ISOOppm
199
22%
29*%
ISOOOppm
FO males and females
Increased absolute and relative liver weights were observed in FO
males (>1,500 ppm) and females (15,000 ppm) (data not provided)
Absolute liver weight Fl weanlings
M 0% 5%
F 0% 6%
12*%
17*%
20*%
21*%
Liver/body weight Fl weanlings
M 0% 0%
F 0% 0%
10*%
10*%
30*%
33*%
Absolute liver weight Fl adults
M 0% -2%
F 0% 6%
5%
6%
14*%
15*%
Liver/body weight Fl adults
M 0% 2%
3%
18*%
This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                     A-15            DRAFT—DO NOT CITE OR QUOTE

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                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design





Saegusa et al. (2009)
Crj:CD(SD)IGS rats, 10 dams/group,
litters culled to 4/sex/dam on PND 2, Fl
animals maintained for 11 wks
(10/sex/group for liver weight)
0, 100, 1,000, 10,000 ppm (TWAC: 0,
14.8, 146.3, 1,505 mg/kg-d)
Diet
GD10-PND20
Liver chemistry
van derVen et al. (2006)
Wistar rats, 3-5/sex/group for liver
chemistry
On 3 1 3 in 3n inn ~>nn mn/bn H
Gavage
zo u

van derVen et al. (2009)
Wistar rats, 10/sex/group, Fl offspring
evaluated at PND 21 (2/sex/litter) and
PNW 11 (5/sex/group)
0, 0.1, 0.3, 1, 3, 10, 30, 100 mg/kg-d
Diet
One full spermatogenic or two full
estrous cycles (males: 70 d prior to
mating; females: 14 d prior to mating)
and continued during pregnancy and
lactation for a total of 11 wks post
weaning
Results
F 0% 5%
Absolute liver weight F2 weanlings
M 0% 4%
F 0% 1%
Liver/body weight F2 weanlings
M 0% 0%
F 0% 0%
Percent change compared to control
Doses 0 14.8
Liver/body weight: F1(PND20)
M 0% 4%
F 0% 2%
Liver/body weight: Fl (wk 11)
M 0% 10*%
F 0% 7%

Percent change compared to control
Doses 0 0.3 1 3
T4-UGTf
M 0% 22% 11% 92%
F 0% 9% -5% -23%
Sum of apolar liver retinoids
M 0% 44% 21% 42%
F 0% 1% -13% -12%
Percent change compared to control
Doses 0 0.1 0.3 1
Sum of apolar liver retinoids
M 0% 7% 14% 32%
F 0% 30% 27% 38%


5%

6%
2%

7%
5%

146.3

8%
6%

4%
3%


10 30

67% 103%
2% 32%

19% 16%
-26% -21%

3 10

-4% -5%
2% 0%


21*%

0%
-4%

27*%
25*%

1505

27*%
28*%

2%
-1%


100 200

175% 144%
148% 77%

-5% 22%
-7% -15%

30 100

-4% -19%
9% -17%

* Statistically significantly different from the control at p < 0.05, ** indicates p<0.01
! Doses were based on standard values for body weight and food consumption in BSCSFj mice in a chronic study [i.e., average
 male and female body weight = 0.0363 kg and food consumption = 0.00625 kg/day; U.S. EPA (1988)1.
3 Pairwise significance tests were conducted by EPA. For incidence data, Fisher's Exact tests were used. All statistical analyses
      This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                 A-16              DRAFT—DO NOT CITE OR QUOTE

-------
                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD
      Reference and Study Design                                      Results
were conducted using the freely available R statistical software (version 3.0.1).  For continuous data (where means and
standard deviations are provided), Student's T-tests were used.
CTWA doses were estimated based on food intake and body weight data (as reported by study authors).
 Percent change compared to control calculated as: (treated value - control value)/control value x 100.
e Quality of only available copy of report was difficult to read; values in tables could not be verified with certainty.
'significant dose response as reported by authors.
GD = gestation day; PND = postnatal day; PNW = postnatal week; T4-UGT = hepatic T4-UDP (uridine diphosphate)
 glucuronosyltransferase; TWA = time-weighted average
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                   A-17              DRAFT—DO NOT CITE OR QUOTE

-------
                                                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD
I

2
• Statistically Significant O Not Statistically Significant
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** Significant dose response as reported by study authors
Figure A-2. Exposure-response array of liver effects following oral exposure to HBCD
                          This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                               A-18                             DRAFT—DO NOT CITE OR QUOTE

-------
                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
1   A.3.3. Neurological Effects Evidence Table and Exposure-response Array
2
3
Table A-4. Evidence pertaining to neurological effects in animals following
oral exposure to HBCD
Reference and Study Design
Results"
Neurobehavior
(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGSBRrats, 20-
40/sex/group
0, 100, 300, 1,000 mg/kg-d
Gavage
90 d
(WIL Research Labs (1998), 1997))
Crl:CD(SD)BR rats, 6-12/sex/group
0, 125, 350, 1,000 mg/kg-d
Gavage
28 d
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group; Fl
and F2 offspring produced, Fl
generation neurobehavior endpoints,
10/sex/group
0, 150, 1,500, 15,000 ppm (mean daily
intakes):
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
Diet
10 wks prior to mating and through
gestation, lactation, and for two
generations (multi-generation
reproductive toxicity study)
No treatment-related effects were observed following FOB (home cage,
handling, open field, sensory, neuromuscular, or physiological
observations).
No treatment-related effects were observed following FOB (home cage,
handling, open field, sensory, neuromuscular, or physiological
observations).
Percent change compared to control"
Doses Oppm ISOppm
ISOOppm ISOOOppm
Surface righting reflex response time Fl pups
M 0% -13%
F 0% -23%
-22% -30*%
-7% -16%
Mid-air righting reflex completion rate Fl pups
M 0% 0%
F 0% 0%
0% 0%
0% 0%
Surface righting reflex response time F2 pups
M 0% -5%
F 0% 4%
33% 5%
-9% 61%
Mid-air righting reflex completion rate F2 pups
M 0% 0%
F 0% 0%
-6% 0%
-10% -23*%
Negative geotaxis reflex
M There was no exposure effect in either generation
F
Spontaneous motor activity (Fl males and females)
No significant difference between control and
4 wks of age.
HBCD-treated groups at
T-maze swim test (Fl males and females)
         This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                              A-19           DRAFT—DO NOT CITE OR QUOTE

-------
                               Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design

Eriksson et al. (2006)
NMRI mice, 10-17 males/group (3-
4 litters/dose group)
0, 0.9, 13.5 mg/kg
Single-dose gavage
PND10
Results3
Testing was performed at 6 weeks of age. A straight channel swim test
on day 1 showed no difference in swim speed. Swim maze testing was
performed on days 2-4.
Male rats in the middle- and high-dose groups demonstrated
statistically significant shorter elapsed time as compared to controls on
day 3 of testing, but not on day 2 or 4; male rats in the high-dose group
made fewer errors than control animals on day 3 of testing (but not on
day 2 or 4). Female rats demonstrated no significant difference
between controls and treated rats.
Spontaneous motor activity at 3 mo (n = 10/dose group):
Doses
0 mg/kg 0.9 mg/kg 13.5 mg/kg
Locomotion
0-20 min
20-40 min
40-60 min
Rearing
0-20 min
20-40 min
40-60 min
Total activity
0-20 min
20-40 min
40-60 min
J,* 4,**
_ _ /K**

J,* vj,**
_ _ /K**

4,**
_ _ /K**
Morris water maze at 3 mo (10-12/dose group):
Increased latency to find hidden platform (d 4) and increased
time to find new platform location (d 5) at 13.5 kg/d
Electrophysiological changes
Lilienthal et al. (2009b)
Wistar rats, 3-5/sex/group
0, 0.1, 0.3, 1, 3, 10, 30, 100 mg/kg-d
Diet
One full spermatogenic or two full
estrous cycles (males: 70 d prior to
mating; females: 14 d prior to mating)
and continued during pregnancy and
lactation for a total of 11 wks post
weaning
Percent change compared to control"
Doses 0
0.1 0.3 1 3 10 30 100
Latency of foreleg on catalepsy test
F1M 0% 11% -22% -27% -4% 4% -27% -49%
F1F 0% -44% -6% 7% -19% -53% -59% -56%
BAEP thresholds following stimulation with click
Fl M 0%
Fl F 0%
-3% -44% 9% 0% 0% 29% 47%
7% 21% 18% -7% 23% 11% 9%
* Statistically significantly different from the control at p < 0.05, ** indicates p < 0.01
a Percent change compared to control calculated as: (treated value - control value)/control value x 100.
FOB = functional observational battery; GD = gestation day; PND = postnatal day; PNW = postnatal week;
 TWA = time-weighted average
     This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                              A-20             DRAFT—DO NOT CITE OR QUOTE

-------
                                                                            Preliminary Materials for the IRIS Toxicological Review ofHBCD
l

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-------
                                Preliminary Materials for the IRIS Toxicological Review ofHBCD
1   A.3.4. Developmental Effects Evidence Table and Exposure-response Array
2
3
Table A-5. Evidence pertaining to developmental effects in animals following
oral exposure to HBCD
Reference and Study Design
Results
Development
van derVen et al. (2009)
Wistar rats, 10/sex/dose, 4-9 Fl litters/group
evaluated for developmental effects
0, 0.1, 0.3, 1, 3, 10, 30, 100 mg/kg-d
Diet
One full spermatogenic or two full estrous cycles
(males: 70 d prior to mating; females: 14 d prior
to mating) and continued during pregnancy and
lactation for a total of 11 wks post weaning
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and F2
offspring produced; 18-24 litters/group
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
Diet
10 wks prior to mating and through gestation,
lactation, and for two generations (multi-
generation reproductive toxicity study)
Percent change compared to control"
Doses 0 0.1 0.3 1 3 10
30 100
Time to vaginal opening Fl offspring
Fb 0% 0% 2% 4% 4% 0%
-2% 13%
Anogenital distance (PND 4)
Mb 0% 11% 2% 4% 9% 9%
F 0% 6% -3% 0% 0% 6%
-2% 17%
0% 3%
PND 7 and 21: unaltered
Preputial separation
No exposure-related effect in male Fl pups
Percent change compared to control"
Doses Oppm ISOppm ISOOppm
ISOOOppm
Viability index during lactation FO parents/Fl offspring
DO
D4
D21
99.6% 97.5% 98.8%
95.6% 98.7% 98.7%
93.2% 99.4% 98.1%
99.2%
95.8%
93.8%
Viability index during lactation Fl parents/F2 offspring
DO
D4
D21
98.6% 97.7% 96.0%
86.9% 87.3% 92.1%
85.0% 89.6% 71.3%
97.8%
68.4*%
49.7*%
Pup weight during lactation Fl offspring
M(PNDO)
M(PND4)
M(PND7)
M (PND 14
M (PND 21)
F(PNDO)
F(PND4)
F(PND7)
F (PND 14
F (PND 21)
0% 2% 6%
0% 5% 6%
0% 7% 3%
0% 0% 0%
0% 2% 1%
0% 5% 8*%
0% 7% 8%
0% 10% 10%
0% 6% 6%
0% 6% 7%
0%
-7%
-5%
-7%
-9*%
3%
-4%
-2%
-3%
-6%
Pup weight during lactation F2 offspring
M(PNDO)
M(PND4)
0% -1% 4%
0% 2% -1%
-3%
-12%
         This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                             A-22            DRAFT—DO NOT CITE OR QUOTE

-------
                                  Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design

Saegusa et al. (2009)
Crj:CD(SD)IGS rats, 10 dams/group, litters culled
to4/sex/dam on PND2
1,505 mg/kg-d
Diet (soy-free)
GD 10-PND 20 (weaning)
Results
M(PND7)
M(PND14
M(PND21)
F(PNDO)
F(PND4)
F(PND7)
F(PND14
F(PND21)
0% 5% -3% -22*%
0% 8% -1% -23*%
0% 6% 2% -20*%
0% -3% 3% -5%
0% -4% -1% -18*%
0% 0% -6% -25*%
0% 0% -6% -23*%
0% 2% -2% -20*%
Anogenital distance
M(FO)
F (FO)
M(F1)
F (Fl)
0% 1% 0% -3%
0% 3% 1% -1%
0% 0% -2% -5%
0% 1% 1% -6%
Percent change compared to control"
Doses
0 14.8 146.3 1505
Anogenital distance Fl (PND 1)
M
F
0% 2% 5% 3%
0% -9% -6% -5%
Pup weight Fl(PNDl)
M
F
0% 2% 8% 1%
0% 5% 12% 5%
* Statistically significantly different from the control at p < 0.05,  ** indicates p < 0.01
a Percent change compared to control calculated as: (treated value - control value)/control value x 100.
 Significant dose response as reported by authors.
CTWA doses were estimated based on food intake and body weight data (as reported by study authors).
GD = gestation day; PND = postnatal day; TWA = time-weighted average
      This document is a preliminary draft for review purposes only and does not constitute Agency policy,
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                                                                    Preliminary Materials for the IRIS Toxicological Review ofHBCD
f"l f~l f~l j"l ^c*--,i-;^t;^-^,IL,c;^^.;f;^-',r-,t /-\M,-,»-c»-.-,t;.-t;,~-.ll.,c;rt«;f;^.
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              ' Significant dose response as reported by study authors
Figure A-4. Exposure-response array of developmental effects following oral exposure to HBCD
                           This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                              A-24                                DRAFT—DO NOT CITE OR QUOTE

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                                     Preliminary Materials for the IRIS Toxicological Review ofHBCD
1    A.3.5. Reproductive Effects Evidence Table and Exposure-response Array
2
3
Table A-6.  Evidence pertaining to reproductive effects in animals following
oral exposure to HBCD
              Reference and Study Design
                                                            Results
     Female reproduction
     Maranghi et al. (2013)
     BALB/c female mice
     0 (15/group), 199 mg/kg-d (10/group)
     Diet
     28 d
                                                 Percent change compared to control"
                                     Doses
 0
 199
                                    Testosterone (T)
                                    Estradiol (E2)
                                    T/E2 ratio
0%

0%

0%
57*%

 -9%

56*%
     BASF (1990)
     Sprague-Dawley rats, 5/sex/group
     0,1, 2.5, 5.0% (males: 0, 900, 2,400,
     4,700 mg/kg-d; females: 0, 900, 2,300,
     4,900 mg/kg-d)b
     Diet
     28 d
                                     Decreased number of mature and developing follicles in the
                                     ovaries of high-dose group; incidence data were not provided.
     van derVen et al. (2009)
     Wistar rats, 10 FO/sex/group
     0, 0.1, 0.3,1, 3,10, 30,100 mg/kg-d
     Diet
     One full spermatogenic or two full estrous cycles
     (males: 70 d prior to mating; females: 14 d prior
     to mating) and continued during pregnancy and
     lactation for a total of 11 wks post weaning
                                     No exposure related changes in reproductive parameters,
                                     including mating success, time to gestation, gestation
                                     duration, number of implantation sites, litter size, and sex
                                     ratio.
     Saegusa et al. (2009)
     Crj:CD(SD)IGS rats, 10 dams/group, litters culled
     to4/sex/dam on PND2
     0, 100, 1,000, 10,000 ppm (TWAb: 0, 14.8, 146.3,
     1,505 mg/kg-d)
     Diet (soy-free)
     GD  10-PND 20 (weaning)
                                     No exposure-related changes in reproductive parameters,
                                     including gestation length, number of implantation sites, and
                                     number of live offspring.
           This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                   A-25             DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and F2
offspring produced, reproductive endpoints
primordial follicles were only assessed in Fl
females (10/group)
0, 150, 1,500, 15,000 ppm (mean daily intakes):
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
Diet
10 wks prior to mating and through gestation,
lactation, and for two generations (multi-
generation reproductive toxicity study)
Results
Percent change compared to control"
Doses 0 ppm 150 ppm
1500 ppm ISOOOppm
Primordial follicles Fl adults
F 0% -7%
-37*% -36*%
Number of litters totally lost
FO dam no exposure-related effect reported
Fldam 1/23 1/23 0/20 8/21*
Fertility indexc (male/female) FO parents/Fl offspring
M 100%*d 91.7%
F 100%*d 91.7%
90.9% 85.7%
90.9% 86.4%
Fertility indexc (male/female) Fl parents/F2 offspring
M 95.8% 95.8%
F 95.8% 95.8%
Incidence of pregnancy
FO dam 24/24*d 22/24
Fl dam 23/24 23/24
No exposure-related changes were foi
reproductive parameters, including es
copulation index, fertility index pre-co
implantation sites, gestation index, de
length, litter size, or number and sex o
FOor Fl dams.
87.0% 87.5%
87.5% 87.5%

20/24 19/23
21/24 21/24
ind in any other female
trous cyclicity,
tal interval, number of
livery index, gestation
f live and dead pups, in
Male reproduction
(WIL Research Labs (2002), 2001))
Crl:CD(SD)IGS BR rats, 20-40/sex/group
0, 100, 300, 1,000 mg/kg-d
Gavage
90 d
van derVen et al. (2009)
Wistar rats, 10 FO/sex/group, organ weights in Fl
offspring evaluated at PND 21 (2/sex/group) and
wk 11 (5/sex/group), sperm parameters were
Percent change compared to control"
Doses 0 100
300 1000
Absolute prostate weight Fl (wk 13)
0% 4%
18% 32*%
Prostate/body weight Fl (wk 13)
0% 3%
17% 43*%
Absolute testis (L+R) weight Fl (wk 13)
0% 3%
2% -2%
Testis/body weight Fl (wk 13)
0% 2%
2% 7%
Percent change compared to control"
Doses 0 0.1 0.3 1
3 10 30 100
Absolute prostate weight6 Fl (wk 11)
M 0% 11% -14% 11% -14% -12% 2% 36*%
This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                     A-26            DRAFT—DO NOT CITE OR QUOTE

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                                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and Study Design
evaluated at PND 21
0, 0.1, 0.3, 1, 3, 10, 30, 100 mg/kg-d
Diet
One full spermatogenic or two full estrous cycles
(males: 70 d prior to mating; females: 14 d prior
to mating) and continued during pregnancy and
lactation for a total of 11 wks post weaning
Ema et al. (2008)
Crl:CD(SD) rats, 24 FO/sex/group, Fl and F2
offspring produced, organ weights were assessed
in all generations 13— 24/sex/group' sperm
parameters were assessed in FO and Fl adults,
23-24/group
0, 150, 1,500, 15,000 ppm (mean daily intakes):
FO male: 0, 10.2, 101, 1,008 mg/kg-d
FO female: 0, 14.0, 141, 1,363 mg/kg-d
Fl male: 0, 11.4, 115, 1,142 mg/kg-d
Fl female: 0, 14.3, 138, 1,363 mg/kg-d
10 wks prior to mating and through gestation,
lactation, and for two generations (multi-
generation reproductive toxicity study)
Results
Absolute testis (L+R) weight6 Fl (wk
M 0% -3% 2% 6%
11)
-4% -65 -1% 14*%
Fl male pups (PND 21)
Wo exposure-related change in reproductive organ weights
The only exposure-related change in Fl sperm parameters
was a dose-related reduction in the ratio of separate sperm
heads.
Percent change compared to control"
Doses Oppm ISOppm
ISOOppm ISOOOppm
Absolute prostate weight
Fl adults 0% -7%
Fl weanlings 0% 5%
F2 weanlings 0% 4%
-4% -5%
5% -13%
4% -25*%
Absolute testis (L+R) weight
Fl adults 0% -3%
Fl weanlings 0% 13*%
F2 weanlings 0% 7%
No exposure-related changes were
male sperm parameters.
-3% -5%
11% 1%
0% -19%
found in either FO or Fl
      * Statistically significantly different from the control at p < 0.05, ** indicates p < 0.01
      a Percent change compared to control calculated as: (treated value - control value)/control value x 100.
      TWA doses were estimated based on food intake and body weight data (as reported by study authors).
      c Fertility index (%) = (number of animals that impregnated a female or were pregnant/number of animals with successful
      copulation) x 100.
      d Statistically significant trend test (p < 0.05) performed by EPA.
      e Significant dose response as reported by authors.
      GD = gestation day; PND = postnatal day; PNW = postnatal week; TWA = time-weighted average
1
2
           This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                        A-27              DRAFT—DO NOT CITE OR QUOTE

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                                                                       Preliminary Materials for the IRIS Toxicological Review ofHBCD
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Figure A-5. Exposure-response array of reproductive effects following oral exposure to HBCD
                            This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                                 A-28                                DRAFT—DO NOT CITE OR QUOTE

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                                Preliminary Materials for the IRIS Toxicological Review ofHBCD
1   A.3.6. Immune Effects Evidence Table and Exposure-response Array
2
3
Table A-7. Evidence pertaining to immune effects in animals following oral
exposure to HBCD
Reference and study design
Results
Immune Effects
Watanabeetal. (2010)
Mouse, Balb/c, female
6-7 /group
0, 1% ppm
Diet
28 days
Post exposure: 5 day intranasal
infection with 10s plaque forming
units (PFU) respiratory syncytial virus
van derVen et al. (2006)
Rats, Wistar, male and female
5/sex/group
Gavage
28 days
Percent change compared to control"
Pulmonary viral titers
F 0
0%
1%
-6%

Percent change compared to control"
NK cell activity/ spleen
M 0 0.3 1 3 10
0% 0% -10% 3% -13%
30 100 200
-47% -14% 4%
F Not examined
Absolute CD4+ cells/spleen
M 0 0.3 1 3 10
0% 7% -7% -21% -21%
30 100 200
-36% -21% -29%
F Not examined
Absolute NK cells/spleen
M 0 0.3 1 3 10
0% -21% -25% -4% -15%
30 100 200
-44% -40% -46%
F Not examined
Total cells/spleen
M 0 0.3 1 3 10
0% 2% -4% -10% -20%
30 100 200
-39% -24% -27%
F Not examined
Neutrophils in blood
M 0 0.3 1 3 10
0% -7% 44% 34% 29%
30 100 200
11% 67% 12%
F Not examined
Lymphocytes in blood
M 0 0.3 1 3 10
0% 0% -4% -4% -3%
30 100 200
0% -5% -1%
F Not examined
White blood cell count in blood
M 0 0.3 1 3 10
0% 14% 23% 6% -4%
30 100 200
-22% 19% 14%
F Not examined
         This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                             A-29            DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
Reference and study design






van derVen et al. (2009)
Rats Wistar male and female
P generation: 10/sex/group
4 males/group
0, 0.1, 0.3, 1, 3, 10, 30, 100 mg/kg-d
+ standard feed control0
Diet
P generation:
Females 14 days prior to mating and
continued in dams through gestation
Fl generation: exposed via milk and
had access to feed of the dam
Intraperitoneal injection with 2xl09
SRBC at 8 weeks of age; boost 15
days















Results
White blood cell count in bone marrow
M 0 0.3 1 3 10
0% 0% -40% 29% -13%
F Not examined
Relative thymus weight
M 0 0.3 1 3 10
0% 0% 9% 0% -18%
F 0 0.3 1 36 10
0% -27% 0% -13% 13%
Percent change compared to control"
SRBC antibody titers (IgM day 7)
M Std 0 0.1 0.3 1 3
87% 0% 20% -93% 93% 7%
F Not examined
SRBC antibody titers (IgG day 21)b
M Std 0 0.1 0.3 1 3
139% 0% 100% -6% 28% -17%
F Not examined
NK cell activity/ spleen
M Std 0 0.1 0.3 1 3
32% 0% 29% -4% 15% 8%
F Not examined
Absolute CD4+ cells/spleen
M Std 0 0.1 0.3 1 3
-6% 0% 6% -15% -7% -11%
F Not examined
Absolute NK cells/spleen
M Std 0 0.1 0.3 1 3
36% 0% 26% 0% 10% 13%
F Not examined
Total cells/spleen
M Std 0 0.1 0.3 1 3
-2% 0% 10% -8% -4% -10%
F Not examined
Neutrophils in bloodb
M Std 0 0.1 0.3 1 3
13% 0% -5% -4% -8% 8%
F Not examined
Lymphocytes in bloodb
M Std 0 0.1 0.3 1 3
-2% 0% 0% 0% 0% -1%
F Not examined
White blood cell count in bloodb
M Std 0 0.1 0.3 1 3
-4% 0% 41% 12% 27% -4%


30 100 200
-8% 9% -42%


30 100 200
-18% -9% -9%
30 100 200
0% 0% 0%


10 30 100
13% 33% -7%


10 30 100
144% 378% 161%


10 30 100
5% 22% -5%


10 30 100
-4% 16% -25%


10 30 100
13% 33% 15%


10 30 100
0% 18% -12%


10 30 100
3% 12% 43%


10 30 100
0% -1% -4%


10 30 100
16% 29% -20%
This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                     A-30            DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofHBCD
1
2
3
4
5
6
Reference and study design

Results
F
Not examined
White blood cell count in bone marrowb
M
F
Std 0 0.1 0.3 1 3
94%* 0% 61% 83% 40% 94%
10 30
115% 72%
100
94%
Not examined
Absolute thymus weightb
M
F
Std 0 0.1 0.3 1 3
-31%** 0% -13% -15% -10% -19%
Std 0 0.1 0.3 1 3
-16% 0% -16% -18% -14% -2%
10 30
-11% -23%
10 30
-8% -10%
100
-27%
100
-24%
*Statistically significantly different from the control at p < 0.05, ** indicates p < 0.01
a Percent change compared to control calculated as: (treated value - control value)/control value x 100.
b Significant dose response as reported by authors.
Significant differences between the standard feed control and test control were determined by the study authors
This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                     A-31            DRAFT—DO NOT CITE OR QUOTE

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                                                                     Preliminary Materials for the IRIS Toxicological Review ofHBCD

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                                   Preliminary Materials for the IRIS Toxicological Review ofHBCD
1    A.3.7. Information on test material used in experimental animal studies
           Table A-8. Test material information
Study
BASF (1990)
Emaetal. (2008)
Eriksson etal. (2006)
Kurakawa et al. (1984)
Lilienthaletal. (2009a)
Maranghi et al. (2013)
Pharmakologisches Inst (1990b)
Saegusaetal. (2012)
Saegusa et al. (2009)
van derVen etal. (2009)
van derVen etal. (2006)
Watanabe etal. (2010)
(WIL Research Labs (2002),
2001))
(WIL Research Labs (1998),
1997))
Isomeric composition
Composition not reported
a: 5.8%, P: 7.9% y: 83.7%
Composition not reported
Composition not reported
a: 10.28%, P: 8.72%, y: 81.02%
Composition not reported
Composition not reported
Composition not reported
Composition not reported
a: 10.3%, P: 8.7%, y: 81%
a: 10.28%, P: 8.72%, y: 81.01%
Composition not reported
Composition not reported
Composition not reported
Purity
Not reported
99.7% purity
>98% purity
Not reported
Not reported
(noted traces of tetra- and
pentabromocyclododecane)
Not reported
Not reported
>95% purity
>95% purity
Not reported
Not reported
Not reported
Not reported
Not reported
    Note: Because most studies evaluated multiple endpoints and appear in multiple evidence tables, information on
    test materials was not added to the evidence tables to avoid unnecessary repetition.
          This document is a preliminary draft for review purposes only and does not constitute Agency policy,
                                                A-33            DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD


     APPENDIX B.  PRELIMINARY MECHANISTIC STUDY
     INFORMATION
 1          Mechanistic studies (including genotoxicity studies) identified through the literature search
 2   for HBCD (see Figure 2-1, Supporting Studies) are summarized in Table B-l. For each study, this
 3   table provides information on model system and specific assays used, route evaluated, general
 4   target tissues or systems studied, and endpoints reported. The mechanistic studies identified for
 5   HBCD consist largely of in vitro assays; entries for these studies include the cell line origin, identity,
 6   and immortalization/transformation status; culture conditions; and experimental methods.
 7          The information presented in Table B-l illustrates the breadth and scope of the available
 8   mechanistic data for HBCD (e.g., in vivo vs. in vitro, human vs. rodent or non-mammalian system,
 9   and level of organization - organ, system, cellular, or molecular). Mechanistic studies that did not
10   appear to fit into one of these categories were tabulated as "other." Where possible, the following
11   HBCD target descriptors were assigned to each study: endocrine (thyroid, development), hepatic,
12   neurologic, reproduction and development, immunologic, and genotoxic.
13          This table does not include an extraction of detailed study design information (e.g., doses or
14   concentrations, exposure durations) or assay results and, as such, does not represent an evidence
15   table. Identifying the organ or target system will help highlight potential relationships between
16   mechanistic information and toxicity information gathered for characterizing human health
17   hazards related to chronic HBCD exposure.
18
19
          This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                             B-l            DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
        Table B-l. HBCD mechanistic studies
  Reference, effect measured, test system
        Reported endpoints and assays
    Target
Mammalian in vivo
Reistad et al. (2006)

In vivo HBCD distribution

Male Wistar rats (weight from 450 to 550 g);
one single intraperitoneal injection
•  HBCD in rat brain after (IP) injection (brain
   and cerebellum; Analyses of PBDEs by GC-MS)
•  Brain and liver extracts-analyses of HBCD by
   LC-MS
Neurologic,
hepatic,
ADME/PBPK
BASF (2000)
In vivo chromosomal aberrations and
aneuploidy

NMRI mice via i.p. injection
Micronucleus test
Genotoxicity
Mammalian in vitro
AI-Mousa and Michelangeli (2012)
In vitro human neurotoxicity
(neuroblastoma) study

SH-SY5Y human neuroblastoma cells
   Cell Viability Assay [3-(4,5-dimethylthiazol-2-
   yl)-2,5-diphenyltetrazolium bromide (MTT)
   assay; PI and staining and FACs analysis]
   Caspase-3/7 activity (Ac-DEVD-AMC
   fluorescence)
   Cytochrome c Release Assay
   (Immunoblotting)
   Mitochondrial Membrane Potential (Rhl23)
   Reactive Oxygen Species (DCFH-DA)
   Changes in Intracellular [Ca2+] (Fluorescence)
   Ca2+ATPase Activity (phosphate liberation
   assay)
   AP 1-42 level (p-amyloid peptide by ELISA)
Neurologic
Bastos Sales et al. (In Press)

In vitro human neuroblastoma cell viability,
global DNA methylation

Human neuroblastoma (SK-N-AS cells)
   Cell viability [lactate dehydrogenase leakage
   (LDH);3-(4,5-dimethylthiazol-2-yl)-2,5-
   diphenyltetrazolium bromide (MTT) assay]
   Neuroblastoma cells: global DNA methylation
   [(5MdC) as a percentage of the total
   deoxycytidines (dC + 5MdC); arbitrary primed-
   PCR]
Neurologic
Bastos Sales et al. (In Press)

In vitro mouse neuroblastoma cell viability,
global DNA methylation and mouse
preadipocytes differentiation

Mouse neuroblastoma [(Neuro-2A cells
(N2A)]

Mouse preadipocyte
fibroblasts (3T3-L1)
All cells: Cell viability [lactate dehydrogenase
leakage (LDH); 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay]

Mouse neuroblastoma cells: Global DNA
methylation [(5MdC) as a percentage of the total
deoxycytidines (dC + 5MdC); arbitrary primed-
PCR]

Mouse preadipocyte fibroblasts: Cell
differentiation (3T3-L1 differentiation measured
via flow cytometry)
Neurologic
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
     Reported endpoints and assays
    Target
Reistad et al. (2006)

In vitro rat neurotoxicity

Cultured rat cerebellar
granule cells (CGC) from 7-day old pups
Cell viability (Trypan blue exclusion)
Reactive Oxygen Species formation (DCFH-DA)
Changes in Intracellular [Ca2+] (Fluorescence)
Examination of nuclear morphology
(Condensed and fragmented nuclei,
fluorescent probe Hoechst 33258)
Caspase-3/7 activity (Ac-DEVD-AMC
fluorescence)
Internucleosomal DNA fragmentation
(Apoptotic DNA ladder Kit)
Neurologic
Dingemans et al. (2009)
In vitro neuroendocrine rat model

Rat pheochromocytoma (PC12) cells
Cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay]
Changes in Intracellular [Ca2+] (Fluorescence)
Spontaneous and K+-evoked vesicular
catecholamine release (Amperometric
recording)
Neurologic
Mariussen and Fonnum (2003)
In vitro rat neurotoxicity

Rat brain synaptosomes
Synaptosomal uptake of dopamine, glutamate
and GABA (Mariussen and Fonnum, 2001).
Synaptosomal accumulation of 3H-TPP+ as
measure of membrane potential
(Tetra[3H]phenylphosphonium Bromide)
Neurologic
Anetal. (2013)

In vitro human hepatotoxicity study

Immortalized human hepatocyte L02 cell line
Cell survival (Cell Counting kit-8)
Apoptotic cells (TUNEL assay)
Reactive oxygen species (DCFH-DA)
DNA single-strand breakages (comet assay)
Mitochondrial membrane potential (Rhl23)
Changes in Intracellular [Ca2+] (Fluorescence)
Protein expression (Western blot)
Hepatic
Zhang etal. (2008a)

In vitro human hepatotoxicity study

Human hepatoma cells Hep G2 (human
hepatoblastoma cell line)
Cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay]
Cell proliferation determined by total
metabolic activity with resazurine [lactate
dehydrogenase leakage (LDH)]
Reactive Oxygen Species (DCFH-DA)
Hepatic
Huetal. (2009b)

In vitro human hepatotoxicity study

Human hepatoma cells Hep G2 (human
hepatoblastoma cell line)
Cell viability [lactate dehydrogenase leakage
(LDH)]
Morphological observation (inverted
fluorescence microscopy)
Nitric oxide synthase activity (kit)
Intra- or extracellular occurrence of nitrite
(NO2~) (Ding method)
Reactive Oxygen Species (DCFH-DA)
Mitochondrial Membrane Potential (Rhl23)
Hepatic
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
     Reported endpoints and assays
    Target
Canton etal. (2006)
In vitro human adrenocortical carcinoma
CYP17 activity

H295R human adrenocortical carcinoma cell
line
Cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay]
Combined 17a-hydroxylase and 17,20-lyase
activities of CYP17 (DHEA production by
radioimmunoassay)
Endocrine
Kang etal. (2012)
In vitro human endocrine disruption

Human BG-1 ovarian adenocarcinoma cell
line (estrogen-dependent cell line expressing
ERs, including ERa and ERP)
Cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay]
Gene expression (ovarian adenocarcinoma
cells mRNA expression): Real-time PCR: p21,
CDK2, cyclin Dl and GAPDH
Protein expression (Western blot): p21 and
cyclin Dl
Endocrine,
Reproduction
and
Development
Fa et al. (2013)
In vitro toxicity and gene expression in rat
Leydig cells

Primary cultures of Leydig cells obtained
from 51 days old Wistar rats
Cell viability (sulforhodamine B assay)
Change in the mitochondrial membrane
potential (TMRE)
Androgen and progesterone levels in the
collected incubation medium
(radioimmunoassay)
cAMP and cGMP accumulation in collected
media (EIA Kit)
Gene expression (Leydig cell mRNA
expression): Real-time PCR (Kit): receptor Bl
(Scarb-1), steroidogenic factor 1 (Sf-1),
androgen receptor
(Ar) and 3-hydroxysteroid dehydrogenase 1/2
(Hsd3bl/2), cyclooxygenase 2 (Cox-2), LH
receptor (Lhr), translocator protein (Tspo),
steroidogenic acute regulatory protein (Star),
cholesterol side chain cleavage enzyme
(Cypllal), 17-hydroxylase/C17-20-lyase
(Cypl7al), 17-hydroxysteroid dehydrogenase
3 (Hsdl7b3) and dosage-sensitive sex
reversal, adrenal hypoplasia critical region, on
chromosome X, gene 1 (Dax-1), Scarb-1, Star,
Sf-1, Ar, Cypllal and Hsd3bl/2
Protein expression (Western blot); 30 kDa
form of STAR
Endocrine,
Reproduction
and
Development
Park etal. (2012)
In vitro human endocrine disruption

Human BG-1 ovarian adenocarcinoma cell
line (estrogen-dependent cell line expressing
ERs, including ERa and ERP)
Cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay]
Gene expression (ovarian adenocarcinoma
cells mRNA expression): Real-time PCR: Cyclin
D, cdk-4, p21 and GAPDH
Endocrine,
Reproduction
and
Development
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                 B-4               DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
        Reported endpoints and assays
    Target
Yamada-Okabe et al. (2005)
In vitro human cells that over-express
thyroid receptor or estrogen receptor to
monitor endocrine disruption

Human HeLaTR cells that constitutively over-
express human thyroid hormone receptor al

MCF7 (human breast adenocarcinoma) cells
that express human estrogen receptor a
•  Cell viability (MTS)
•  TR and/or ER-mediated gene expression
   (increased luciferase activity)
Endocrine,
Reproduction
and
Development,
Thyroid
Schriksetal. (2006a)

In vitro thyroid hormone disruption in rat

Rat pituitary tumor GH3 cell line [specifically
proliferates when exposed to 3,3',5-triiodo-
L-thyronine (T3)]
•  T-screen (Cell proliferation determined by
   total metabolic activity of GH3 cells with
   resazurine)
•  BrdU-cell proliferation assay (kit)
Endocrine,
Thyroid
Hinkson and Whalen (2009)
In vitro human immune
defense (viral  and tumor)

Human NK Cells isolated from
Peripheral blood from healthy adult (male
and female)
•  Cell viability (trypan blue exclusion)
•  NK cell ability to lyse tumor cells (Cr release
   assay)
•  ATP Assay (Fluorescence)
Immunologic
Hinkson and Whalen (2010)
In vitro human immune defense (viral and
tumor)

Human NK Cells isolated from
Peripheral blood from healthy adult (male
and female)
   Cell viability (trypan blue exclusion)
   Conjugation assay: target cells (NK-
   susceptible K562 cell (human chronic
   myelogenous leukemia) with bound NK cells
   Cell-surface Protein Expression (FACSCalibur
   flow cytometer) for antibodies: anti-CD2,
   CDlla, CDllc, CD16, CD18, CD56, TNF-a and
   Fas-L, monoclonal antibody (mouselgGK
   specific for the human cell surface  protein)
Immunologic
Koike etal. (2012)
In vitro mouse immunotoxicity (splenocyte
and bone marrow cytokine production and
phenotype)

Splenocytes and bone marrow (BM) cells
prepared from atopic prone NC/Nga TndCrlJ
male mice
   Cell viability (WST-1 addition)
   FACS Analysis (expression of cell surface
   molecules via antibodies and fluorescence)
   Quantitation of Cytokines in Culture
   Supernatants: Interferon (IFN)-g, interleukin
   (IL)-4, IL-17, and IL-18 (splenocyte culture
   supernatants); thymus- and activation-
   regulated chemokine, macrophage-derived
   chemokine and IL-12p40 levels (BMDC culture
   supernatants)
Immunologic
Microbiological Associates (1996)

In vitro DNA single- and double-strand
breaks

Human peripheral blood lymphocytes
Chromosomal aberration test
Genotoxicity
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                 B-5              DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
        Reported endpoints and assays
                                                                                            Target
Helledayetal. (1999)
In vitro gene recombination

Chinese hamster ovary (CHO) V79 Sp5 and
SDP8 clones with a spontaneous partial
duplication of the hprt gene
Intragenic recombination (reversion assay)
                                                                                        Genotoxicity
Ethyl Corporation (1990)

In vitro DNA damage

Primary hepatocytes from male F344 rats
                                          Unscheduled DNA synthesis
                                              Genotoxicity
Non-mammalian in vivo
Aniagu etal. (2008)
In vivo hepatotoxicity/genotoxicity model
system in fish

Three-spine stickleback (Gasterosteus
aculeatus)
•  Hepatic global DNA methylation
                                                                                        Hepatic
Zhang etal. (2008b)

In vivo sub-lethal toxicity in fish

Chinese rare minnow (Gobiocypris rarus) (4-
6-month-old)
   Mortality
   Liver: CYP1A1 (ethoxyresorufin-O-deethylase,
   EROD) and CYP2B1 (pentaoxyresorufin-O-
   depentylase, PROD) activities (Burke and
   Mayer)
   Brain:  Reactive Oxygen Species (DCFH-DA),
   lipid peroxidation products (thiobarbituric
   acid-reactive substances, TBARS), protein
   oxidation (protein carbonyl), as well as
   superoxide dismutase (SOD) activity
   (Diagnostic Reagent Kit) and glutathione
   (GSH) content [(5,5-dithiobis-(2-nitrobenzoic
   acid) (DTNB)-oxidized GSH (glutathione
   disulfide, GSSG) recycling assay]
   Blood: DNA damage (Comet assay)
   Whole fish:  Content of HBCD
                                                                                        Hepatic,
                                                                                        Neurologic
Crump et al. (2010)

In vivo exposed chick embryo liver gene
expression

Unincubated chicken (G. Callus domesticus)
eggs - exposure before hatching (prior to
embryogenesis)
   Embryo viability (pipping success)
   HBCD Hepatic and Cerebral Cortical Tissue
   (ng/g ww) concentrations
   Gene expression (Hepatic mRNA expression):
   Real-time RT-PCR [kit: gene targets: b-actin,
   CYP2H1, CYP3A37, UGT1A9, L-FABP,
   deiodinase 2 (DI2), insulin-like growth factor-1
   (IGF-1)]
                                                                                        Development,
                                                                                        Hepatic
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                 B-6               DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
        Reported endpoints and assays
    Target
Deng et al. (2009)

In vivo exposed zebrafish embryo toxicity
and gene expression

Wild-type (AB strain) zebrafish whole
embryos
Hatching success:
•  Embryo malformation (pericardial edema and
   axial spinal curvature)
•  Mortality (missing heartbeat, failure to
   develop somites, and a non-detached tail)
•  Larval length

For successful hatchlings:
•  Embryo cell apoptosis (AO staining)
•  Reactive Oxygen Species (DCFH-DA)
•  Gene expression (Whole embryo mRNA
   expression): Real-time PCR [Kit: gene targets:
   p53, Mdm2, Puma, Bax, Bcl-2, Apaf-1,
   caspase-3, and caspase-9]
•  Caspase-3 and caspase-9 activity (colorimetric
   assay)
Development
Du et al. (2012)

In vivo exposed zebrafish embryo toxicity

Wild-type (AB strain) zebrafish whole
embryos
Hatching success:
•  Mortality (missing heartbeat, coagulation of
   the embryos, a non-detached tail and failure
   to develop somites)
•  Developmental effects (heart rate, hatching
   success, growth of the larvae, survival and
   malformation)

For successful hatchlings:
•  Reactive Oxygen Species (DCFH-DA)
•  Caspase-3 and caspase-9 activity (colorimetric
   assay)
Development
Huetal. (2009a)

In vivo exposed zebrafish embryo toxicity

Wild-type (AB strain) zebrafish whole
embryos
   Mortality (Malformation and death)
   Total protein concentration of zebrafish
   embryo (Bradford method)
   Antioxidant Enzymes and Lipid Peroxidation
   [whole embryo, SOD activities and
   malondialdehyde (MDA) Contents, LPO (thio-
   barbituric assay for MDA)]
   Heat shock protein (Hsp70 levels via Western
   Blot)
Development
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                B-7              DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
     Reported endpoints and assays
    Target
Wu et al. (2013)

In vivo zebrafish embryo cardiac
development

Wild-type (TU strain) zebrafish whole
embryos
Survival rate, whole malformation rate, and
hatching rate
Morphological deformities (cardiac
abnormalities, spinal deformity, altered axial
curvature, and tail malformation)
Cardiac functions (arrhythmia via interbeat
variability): end-diastolic volume (EDV), end-
systolic volume (ESV), stroke volume (SV), and
cardiac output (CO)
Apoptosis (Acridine orange (AO) staining and
caspase-3 activity measurement)
Gene expression (whole zebrafish embryos):
Real-time  PCR: Brilliant SYBR Green QPCR
reagent kit
Development
Palace et al. (2008)

In vivo juvenile rainbow trout endocrine
disruption

Juvenile rainbow trout (Oncorhynchus
my kiss) in vivo exposure
Mortality and Fish growth rates
Liver somatic index (LSI) (liver weight as
percentage of whole body weight)
Liver microsomal phase I [ethoxyresorufin-O-
deethylase (EROD)] and II (UDPGT)
biotransformation enzyme activities
Thyroid axis disruption [Free triiodothyronine
(T3) and thyroxine (T4) in plasma]
Fish deiodinase activity (corresponding
roughly to the  Dl, D2 and D3 activities in
mammals); T4  outer ring deiodination
Thyroid Histopathology: Thyroid epithelial cell
heights
Endocrine,
Development
Palace et al. (2010)

In vivo juvenile rainbow trout endocrine
disruption

Juvenile rainbow trout (Oncorhynchus
my kiss) in vivo exposure
Mortality, fish weight, length or condition
Accumulation of 1 nCi of [125I]-T4 muscle, as
well as the gallbladder containing bile, thyroid
gland (sampled as the entire lower jaw
region), intestine (from stomach to vent),
viscera (included stomach, adipose, spleen,
gonad, pancreas), liver and whole blood
Deiodinase type I and II activities in individual
liver microsomes
Endocrine,
Development
Ronisz et al. (2004)

In vivo juvenile rainbow trout endocrine
disruption and other biomarkers

Juvenile rainbow trout (Oncorhynchus
mykiss) injected via i.p.

Feral eelpout (Zoarces viviparus) 5 days in
vivo experiment (data not shown)
Liver microsomes and cytosol: glutathione-S-
transferase (GST), glutathione reductase (GR)
and catalase
Vitellogenin (VTG) induction in male fish
plasma via ELISA (yolk-precursor produced in
female fish in response to 17-estradiol, i.e.,
endocrine disruption biomarker)
DNA adduct formation (32P-postlabelling
analysis)
Liver somatic index (LSI) (liver weight as
percentage of whole body weight)
Protein expression (Western blot): (PMP70
and rainbow trout only)
Endocrine,
Development,
Genotoxic
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                 B-8              DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
                                                  Reported endpoints and assays
    Target
Zhang etal. (2013)

In vivo identification and expression of
differentially expressed genes (immune
and detoxification defense) in clams

Clam gill (Venerupis philippinarum) in vivo
exposure (in seawater)
                                          Gene expression (Clam gill):
                                          Real-time PCR: genes were chosen for further
                                          study with q-PCR based on novelty, relation with
                                          immunity process and detoxification process
                                          (NADH dehydrogenase subunit 1; Cytochrome c
                                          oxidase subunit 1;  Purine nucleoside
                                          phosphorylase; Hemocyanin subunit 2; C-type
                                          lectin 3; Ferritin; Catalase; Elongation factor 1-
                                          alpha; Dihydrodiol dehydrogenase)
Immunologic
Non-mammalian in vitro
Crump et al. (2008)

In vitro chick embryo liver cells, gene
expression

Cultured chicken embryonic
hepatocytes (CEHs) - exposure after
hatching
                                             Cell viability [Calcein-acetoxymethylester
                                             (AM) assay]
                                             Total RNA (TRIzol reagent Kit)
                                             cDNA synthesis (Superscript II kit)
                                             Gene Expression (hepatic mRNA expression):
                                             Real-time RT-PCR [kit: gene targets: b-actin,
                                             CXR, CYP2H1, CYP3A37, UGT1A9, TR-a, TTR,
                                             deiodinase (Dl) 1, 2, and 3, myelin basic
                                             protein (MBP), THRSP14-a, and L-FABP]
Development
Kling and Forlin (2009)

In vitro Zebrafish liver cell proteomic
analyses

Zebrafish liver (ZFL) cell test system
                                             Cell viability [lactate dehydrogenase leakage
                                             (LDH)]
                                             Two-dimensional gel electrophoresis of
                                             extracted proteins from ZFL cells (63
                                             significant responses)
Development
                                          7-day exposure of tails:
                                          •  Negative effects (fungal infections)
                                          •  Tail regression
Schriksetal. (2006b)

In vitro thyroid hormone disruption

Xenopus laevis tadpole
tail tip regression (regression induced by
3,3',5-triiodo-L-thyronine (T3) exposure) in
premetamorphic tadpoles (developmental
stage 52-53)
Endocrine,
Development
Pharmakologisches Inst (1990a)

Salmonella typhimuhum TA98, TA100,
TA1537
                                          Gene mutation
Genotoxicity
Industrial Bio-Test Laboratories (1990)

S. typhimurium TA98, TA100, TA1535,
TA1537, TA1538
                                          Gene mutation
Genotoxicity
Huntingdon Research Centre (1990)

S. typhimurium TA98, TA100, TA1535
                                          Gene mutation
Genotoxicity
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                 B-9               DRAFT—DO NOT CITE OR QUOTE

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                                 Preliminary Materials for the IRIS Toxicological Review ofHBCD
  Reference, effect measured, test system
        Reported endpoints and assays
    Target
Zeigeretal. (1987)

S. typhimurium TA98, TA100, TA1535,
TA1537, TA1538
Gene mutation
Genotoxicity
SRI International (1990)

S. typhimurium TA98, TA100, TA1535,
TA1537, TA1538
Gene mutation
Genotoxicity
Ogaswara et al. (1983)

S. typhimurium TA92, TA94, TA98, TA100,
TA1535, TA1537
Gene mutation
Genotoxicity
Ethyl Corporation (1990a)

S. typhimurium TA98, TA100, TA1535,
TA1537
Gene mutation
Genotoxicity
Litton Bionetics(1990)

S. typhimurium TA98, TA100, TA1535,
TA1537, TA1538

Saccharomyces cerevisiae D4
Gene mutation
Genotoxicity
Other
Sakaietal. (2009)

In vitro assay for CAR ligand activity in Baikal
seal

Baikal seal (Pusa
sibirica) in vitro reporter
gene assay (Comparison to mouse)
Ligand-dependent transcriptional activation of
constitutive active/androstane receptor (CAR)
Potency (CAR cDNA clones from the Baikal seal
and mouse used for in vitro reporter gene assay)
Undetermined
Schriksetal. (2007)

In vitro thyroid hormone dysruption in
reporter gene assays (monkey cells)

Transient transfection assays; Green monkey
kidney fibroblast
(CV-1) cells transiently transfected with
Xenopus TRs and a luciferase reporter
(TRa/p-specific reporter gene assays)
•  Cell viability (assumed from prior assays)
•  Effects on T3 (ECSO)-induced activation of TRs
Thyroid
      This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                               B-10              DRAFT—DO NOT CITE OR QUOTE

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                                       Preliminary Materials for the IRIS Toxicological Review ofHBCD
       Reference, effect measured, test system
        Reported endpoints and assays
    Target
     Ibhazehieboetal. (2011)
     In vitro neurotoxicity in reporter gene assays
     and newborn rat cultures

     Transient transfection-based
     reporter gene assays [Green monkey kidney
     fibroblast
     (CV-1) cells]

     Interaction of Thyroid hormone receptor
     with Thyroid  hormone
     response element (TRE)

     Purkinje cells in primary cerebellar culture
     derived from newborn rat
   Cell viability (CV-1 cells, Trypan blue
   exclusion)
   TR-mediated transcription using the transient
   transfection-based reporter gene assay in CV-
   1 cells
   TR binding to TRE (liquid chemiluminescent
   DNA pull down assay in vitro
   TH-induced dendrite arborization of Purkinje
   cells
Neurologic,
Endocrine,
Thyroid
     Harju et al. (2007)

     Quantitative structure-activity relationships
     (QSARs) based on in vitro potencies
   Basis: In vitro activities (e.g., chemically
   activated luciferase expression reporter gene
   assay): Androgen, progesterone, estrogen,
   and dioxin (aryl hydrocarbon) receptors, plus
   competition with thyroxine for its plasma
   carrier protein (transthyretin), inhibition of
   estradiol sulfation via sulfotransferase, and
   rate of metabolization
   Physicochemical parameters:  Frontier
   molecular orbitals, molecular charges,
   polarities, log octanol/water partitioning
   coefficient, and two- and three-dimensional
   molecular properties
   Experimental properties: Individual
   ultraviolet spectra (200-320 nm) and
   retention times on three different high-
   performance liquid chromatography columns
   and one nonpolar gas chromatography
   column
QSAR
predictions
     Fernandez Canton et al. (2005)

     Human adrenocortical
     carcinoma cell line (H295R)
Abstract only
Abstract only
1

2
           This document is a preliminary draft for review purposes only and does not constitute Agency policy.
                                                      B-ll             DRAFT—DO NOT CITE OR QUOTE

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