BACKGROUND AND INTRODUCTION

Polybrominated diphenyl ethers (PBDEs) are synthetic brominated flame retardants that are ubiquitous environmental contaminants that have been measured in animals and in humans. Because the developing organism has been shown to be particularly vulnerable to endocrine-disrupting chemicals, such as PBDEs, the committee decided to focus on studies of developmental exposure. PBDEs have been linked to effects on neurodevelopment after developmental exposure in animal studies.

OBJECTIVE AND SPECIFIC AIMS

METHODS

Risk of Bias (Quality) Assessment of Individual Studies

Risk of bias is related to the internal validity of a study and reflects study-design characteristics that can introduce a systematic error (or deviation from the true effect) that might affect the magnitude and even the direction of the apparent effect. Internal validity or risk of bias will be assessed for individual studies using a tool developed by the National Toxicology Program's Office of Health Assessment and Translation (OHAT) that outlines an approach to evaluating risk of bias for experimental animal studies. The risk of bias domains and questions for experimental animal studies are based on established guidance for experimental human studies (randomized clinical trials) (Viswanathan et al. 2012, 2013; Sterne et al. 2014; Higgins and Green 2011) and recent tools for animal studies (Hooijmans et al. 2014; Koustas et al. 2014). The risk of bias tool includes a common set of questions (Section E-1e) that are answered based on the specific details of individual studies to develop risk of bias ratings (using the four options: definitely low risk of bias; probably low risk of bias; probably high risk of bias; or definitely high risk of bias). Information or study procedures that were not reported are assumed not to have been conducted, resulting in an assessment of “probably high” risk of bias. Study design determines the subset of questions that should be used to assess risk of bias for an individual study (see Table E1-1).

Studies are independently assessed by two assessors (BH, SSc) who answer all applicable risk of bias questions with one of four options (see Table E1-2) following prespecified criteria detailed in Section E-1e. The criteria describe aspects of study design, conduct, and reporting required to reach risk of bias ratings for each question and specify factors that can distinguish among ratings (e.g., what separates “definitely low” from “probably low” risk of bias). The instructions and detailed criteria are tailored to the specific type of human study designs. Risk of bias will be assessed at the outcome level because study-design or method specifics may increase the risk of bias for some outcomes and not others within the same study. Information or study procedures that were not reported are assumed not to have been conducted, resulting in an assessment of “probably high” risk of bias. Authors will be queried by email to obtain missing information, and responses received will be used to update risk of bias ratings.

Assessors will be trained using the criteria in an initial pilot phase undertaken to improve clarity of criteria that distinguish between adjacent ratings and to improve consistency among assessors. All team members involved in the risk of bias assessment will be trained on the same set of studies and asked to identify potential ambiguities in the criteria used to assign ratings for each question. Any ambiguities and rating conflicts will be discussed relative to opportunities to refine the criteria to more clearly distinguish between adjacent ratings. If major changes to the risk of bias criteria are made based on the pilot phase (i.e., those that would likely result in revision of response), they will be documented in a protocol amendment along with the date modifications were made and the logic for the changes. It is also expected that information about confounding, exposure characterization, outcome assessment, and other important issues may be identified during or after data extraction, which can lead to further refinement of the risk of bias criteria.

After assessors have independently made risk of bias determinations for a study across all risk of bias questions, the two assessors will compare their results to identify discrepancies and attempt to resolve them. Any remaining discrepancies will be considered and resolved with the review team. The final risk of bias rating for each question will be recorded along with a statement of the basis for that rating.

Data Analysis and Evidence Synthesis

The review team will qualitatively synthesize the body of evidence for each outcome and, where appropriate, a meta-analysis will be performed. If a meta-analysis is performed, summaries of main characteristics for each included study will be compiled and reviewed by two team members to determine comparability between studies, to identify data transformations necessary to ensure comparability, and to determine whether heterogeneity is a concern. The main characteristics considered across all eligible studies include the following:

• Experimental design (e.g., acute, chronic, multigenerational)
• Animal model used (e.g., species, strain, sex, genetic background)
• Age of animals (e.g., at start of treatment, mating, and/or pregnancy status)
• Developmental stage of animals at treatment and outcome assessment
• Dose levels, frequency of treatment, timing, duration, and exposure route
• Health outcome(s) reported
• Type of data (e.g., continuous or dichotomous), statistics presented in paper, access to raw data
• Variation in degree of risk of bias at individual study level

TABLE E1-1

OHAT Risk of Bias Tool.

TABLE E1-2

Answers to the Risk of Bias Questions.

The review team expects to require input from subject-matter experts to help assess the heterogeneity of the studies. Subgroup analyses to examine the extent to which risk of bias contributes to heterogeneity will be performed. If a meta-analysis is considered appropriate, the review team will stratify by species and further consider separate meta-analyses by species. Situations where it may not be appropriate to include a study are when data on exposure or outcome are too different to be combined or other circumstances that may indicate that averaging study results would not produce meaningful results. When considering outcome measures for conducting meta-analyses, benchmark dose (BMD) estimates (and their associated confidence intervals) with a benchmark response (BMR) set to a common percent of control (for continuous outcomes) or extra risk (for dichotomous outcomes) are preferred. A secondary alternative, when there are more than two groups, is the conduct of BMD modeling and the use of the derived BMD estimates. Meta-analyses are not possible with lowest-observed-adverse-effect levels or no-observed-adverse-effect levels, since no confidence interval can be derived for these measures.

If a meta-analysis is conducted, a random effects model will be used for the analysis. Heterogeneity will be assessed using the I-squared statistic. Interpretation of I-squared will be based on the Cochrane Handbook: 0% to 40% (might not be important); 30% to 60% (may represent moderate heterogeneity); 50% to 90% (may represent substantial heterogeneity); and 75% to 100% (considerable heterogeneity). Additionally, as described in the Cochrane Handbook, for the last three categories, the importance of the I-squared will be interpreted considering not only the magnitude of effects but also the strength of the evidence (90% two-tailed confidence interval).

The review team will also perform sensitivity analyses on the exclusion of individual studies in succession.

If sufficient studies are available, subgroup analyses will be performed based on the following characteristics described above: experimental design, animal model used (e.g., species and/or strain), age of animals, and developmental stage of animals at treatment and outcome assessment.

In the event that these proposed methods for data analysis are altered to tailor to the evidence base from included studies, the protocol will be amended accordingly, and the reasons for change will be justified in the documentation.

Confidence Rating: Assessment of the Body of Evidence

The quality of evidence for each outcome will be evaluated using the GRADE system for rating the confidence in the body of evidence (Guyatt et al. 2011; Rooney et al. 2014). More detailed guidance on reaching confidence ratings in the body of evidence as “high,” “moderate,” “low,” or “very low” is provided in NTP (2016, see Step 5). In brief, available studies on a particular outcome are initially grouped by key study-design features, and each grouping of studies is given an initial confidence rating by those features.

The initial rating is downgraded for factors that decrease confidence in the results, including

• risk of bias
• unexplained inconsistency
• indirectness or lack of applicability
• imprecision
• publication bias

The initial rating is upgraded for factors that increase confidence in the results, including

• large magnitude of effect
• dose response
• consistency across study designs/populations/animal models or species
• consideration of residual confounding
• other factors that increase confidence in the association or effect (e.g., particularly rare outcomes)

The reasons for downgrading (or upgrading) confidence may not be due to a single domain of the body of evidence. If a decision to downgrade is borderline for two domains, the body of evidence is downgraded once in a single domain to account for both partial concerns based on considering the key drivers of the strengths or weaknesses. Similarly, the body of evidence is not downgraded twice for what is essentially the same limitation (or upgraded twice for the same asset) that could be considered applicable to more than one domain of the body of evidence. Consideration of consistency across study designs, human populations, or animal species is not included in the GRADE guidance (Guyatt et al. 2011); however, it is considered in the modified version of GRADE used by OHAT (Rooney et al. 2014).

Confidence ratings are independently assessed by members of the review team, and discrepancies will be resolved by consensus and consultation with technical advisors as needed. Confidence ratings will be summarized in evidence profile tables.

REFERENCES

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• Higgins J, Green S, editors. Cochrane Handbook for Systematic Reviews of Interventions, Version 5.1.0 (updated March 2011). The Cochrane Collaboration; 2011. [May 6, 2016]. http://handbook​.cocharne.org.
• Hooijmans CR, Rovers MM, de Vries RB, Leenars M, Ritskes-Hoitinga M, Langendam MW. SYRCLE's risk of bias tool for animal studies. BMC Med. Res. Method. 2014;14:43. [PMC free article: PMC4230647] [PubMed: 24667063]
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• NTP (National Toxicology Program). Handbook for Conducting a Literature-Based Health Assessment Using OHAT Approach for Systematic Review and Evidence Integration. Office of Health Assessment and Translation, Division, National Toxicology Program, National Institute of Environmental Health Sciences; Jan 9, 2015. 2015. [September 21, 2015]. http://ntp​.niehs.nih​.gov/ntp/ohat/pubs/handbookjan2015_508​.pdf.
• Robinson KA, Whitlock EP, O'Neil ME, Anderson JK, Hartling L, Dryden DM, Butler M, Newberry SJ, McPheeters M, Berkman ND, Lin JS, Chang S S. Research white paper. Rockville, MD: Agency for Healthcare Research and Quality; 2014. [May 9, 2016]. Integration of Existing Systematic Reviews. https://www​.ncbi.nlm​.nih.gov/books/NBK216379/ AHRQ Publication No. 14-EHC016-EF. [PubMed: 25032273]
• Robinson KA, Chou R, Berkman ND, Newberry SJ, Fu R, Hartling L, Dryden D, Butler M, Foisy M, Anderson J, Motu'apuaka M, Relevo R, Guise JM, Chang S. Methods Guide for Effectiveness and Comparative Effectiveness Reviews. Rockville, MD: Agency for Healthcare Research and Quality; 2015. [May 9, 2016]. Integrating Bodies of Evidence: Existing Systematic Reviews and Primary Studies. https://www​.ncbi.nlm​.nih.gov/books/NBK279904/ AHRQ Publication No. 15-EHC007-EF.
• Robinson KA, Chou R, Berkman ND, Newberry SJ, Fu R, Hartling L, Dryden D, Butler M, Foisy M, Anderson J, Motu'apuaka M, Relevo R, Guise JM, Chang S. Twelve recommendations for integrating existing systematic reviews into new reviews: EPC guidance. J. Clin. Epidemiol. 2016;70:38–44. [PubMed: 26261004]
• Rooney AA, Boyles AL, Wolfe MS, Bucher JR, Thayer KA. Systematic review and evidence integration for literature-based environmental health assessments. Environ. Health Perspect. 2014;122(7):711–718. [PMC free article: PMC4080517] [PubMed: 24755067]
• Sterne JAC, Higgins JPT, Reeves BC. ACROBAT-NRSI: A Cochrane risk of Bias Assessment Tool for Non-randomized Studies of Interventions. Version 1.0.0. Sep 24, 2014. 2014. [May 6, 2016]. www​.riskofbias.info.
• Viswanathan M, Ansari M, Berkman ND, Chang S, Hartling L, McPheeters LM, Santaguida PL, Shamliyan T, Singh K, Tsertsvadze A, Treadwell JR. Assessing the Risk of Bias of Individual Studies when Comparing Medical Interventions. Rockville, MD: Agency for Healthcare Research and Quantitative Methods Guide for Comparative Effectiveness Reviews; 2012. [May 6, 2016]. www​.effectivehealthcare.ahrq.gov/ AHRQ Publication No. 12-EHC047-EF.
• Viswanathan M, Berkman ND, Dryden DM, Hartling L. Methods Research Report. Rockville, MD: Agency for Healthcare Research and Quantitative Methods Guide for Comparative Effectiveness Reviews; 2013. [May 6, 2016]. Assessing Risk of Bias and Confounding in Observational Studies of Interventions or Exposures: Further Development of the RTI Item Bank. www​.effectivehealthcare​.ahrq.gov/reports/final.cfm. AHRQ Publication No. 13-EHC106-EF. [PubMed: 24006553]
• Whiting P, Savovic J, Higgins JB, Caldwell DM, Reeves BC, Shea B, Davies P, Kleijnen J, Churchill R. ROBIS: A new tool to assess risk of bias in systematic reviews was developed. J. Clin. Epidemiol. 2016;69:225–234. [PMC free article: PMC4687950] [PubMed: 26092286]
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Footnotes

1

A systematic review “is a scientific investigation that focuses on a specific question and uses explicit, prespecified scientific methods to identify, select, assess, and summarize the findings of similar but separate studies” (IOM 2011, p. 1).

2

HAWC (Health Assessment Workspace Collaborative): A Modular Web-based Interface to Facilitate Development of Human Health Assessments of Chemicals (https://hawcproject​.org/portal/).

SECTION E-1a. REVIEW TEAM BIOGRAPHICAL INFORMATION

Jaime F. Blanck is a clinical informationist at the Welch Medical Library at Johns Hopkins University. She creates and implements systematic review search strategies across multiple databases and provides comprehensive reference, research, and information services to multiple departments within the School of Medicine. She received an MLIS from the University of Pittsburgh and an MPA from the University of Baltimore.

Barbara F. Hales is a James McGill Professor in the Department of Pharmacology and Therapeutics at McGill University. Her research interests are in the mechanisms of action of drugs as teratogens. She studies developmental toxicity using a combination of in vivo, in vitro, and molecular approaches with the goal of elucidating how the embryo responds to insult after direct or maternal exposure and the consequences to progeny of paternal drug exposure. Dr. Hales is a past president of the Teratology Society, and is currently co-chair of the Chemicals Management Plan Science Committee of the Government of Canada. She received an MSc in pharmacognosy from the Philadelphia College of Pharmacy and Science and a PhD in pharmacology and therapeutics from McGill University.

Ellen Mantus is a scholar and director of risk assessment on the Board on Environmental Studies and Toxicology at the National Academies of Sciences, Engineering, and Medicine with more than 20 years of experience in the fields of toxicology and risk assessment. She has served as the study director on numerous projects, including ones that have assessed the health implications of various chemical exposures; developed strategies for applying modern scientific approaches in toxicology and risk assessment; provided guidance to federal agencies on risk-based decision making; and evaluated barriers to deployment of electric vehicles and associated charging infrastructure. Before joining the National Academies, Dr. Mantus was a project manager with ICF Consulting where she served as a primary reviewer for numerous toxicological studies and provided risk assessment and regulatory support on a wide array of projects. Dr. Mantus received a PhD in chemistry from Cornell University.

Susan Martel is a senior program officer in the Board on Environmental Studies and Toxicology at the National Academies of Sciences, Engineering, and Medicine. She has 20 years of experience in supporting toxicology and risk assessment projects for the US Environmental Protection Agency, the US Department of Defense, and the National Aeronautics and Space Administration. Recent projects include working with committees evaluating the toxicological effect of arsenic, developing exposure guidelines for use on spacecraft, and assessing pesticide risks-assessment practices. Before joining the National Academies, she was the administrator of the Registry for Toxicology Pathology for Animals at the American Registry of Pathology. She received a BA in biology from Skidmore College.

Susan L. Schantz is a professor of toxicology in the Department of Comparative Biosciences, College of Veterinary Medicine, at the University of Illinois at Urbana-Champaign. She is also director of a National Institute of Environmental Health Sciences (NIEHS) T32 training program in endocrine, developmental, and reproductive toxicology and director of a Children's Environmental Health Research Center jointly funded by the NIEHS and the EPA. In addition, she is currently the interim director of the Neuroscience Program. Dr. Schantz's research interests involve understanding the neurobehavioral effects of chemical exposures during development and aging. She conducts research in both laboratory-based animal studies and parallel epidemiologic studies. She has served as president of the Neurotoxicology Specialty Section of the Society of Toxicology and president of the Neurobehavioral Teratology Society. Dr. Schantz was also a member of the NRC's Committee to Assess the Health Implications of Perchlorate Ingestion. She received a PhD in environmental toxicology from the University of Wisconsin–Madison.

SECTION E-1b. LITERATURE SEARCH STRATEGY

The review team will employ a multi-method process to identify all potentially relevant studies as detailed below.

SECTION E-1c. SCREENING FORMS

SECTION E-1d. DATA EXTRACTION ELEMENTS FOR ANIMAL STUDIES

SECTION E-1e. RISK OF BIAS QUESTIONS FOR ANIMAL STUDIES

1. Was administered dose or exposure level adequately randomized?

2. Was allocation to study groups adequately concealed?

3. Did selection of study participants result in the appropriate comparison groups? [NA]

4. Did study design or analysis account for important confounding and modifying variables? [NA]

5. Were experimental conditions identical across study groups?

6. Were the research personnel blinded to the study group during the study?

7. Were outcome data complete without attrition or exclusion from analysis?

8. Can we be confident in the exposure characterization?

9. Can we be confident in the outcome assessment?

10. Were all measured outcomes reported?

11. Was litter or litter effects considered appropriately in the statistical analyses and were there no other potential threats to internal validity?

Because this evaluation is focused on developmental exposure, this question was added to address litter effects in data analysis. This question will be used to examine individual studies for appropriate statistical methods (e.g., confirmation of homogeneity of variance for ANOVA and other statistical tests that require normally distributed data). It will also be used for risk of bias considerations that do not fit under the other questions.

SECTION E-1f. AMENDMENTS TO THE PROTOCOL

• Changes to the Review Team
• (September 15, 2016)

Original review team members Barbara Hales and Susan Schantz were replaced by the following committee members who have more experience conducting risk of bias evaluations and data extraction:

• David C. Dorman (Chair) is a professor of toxicology in the Department of Molecular Biosciences of North Carolina State University. The primary objective of his research is to provide a refined understanding of chemically induced neurotoxicity in laboratory animals that will lead to improved assessment of potential toxicity in humans. Dr. Dorman's research interests include neurotoxicology, nasal toxicology, pharmacokinetics, and cognition and olfaction in animals. He has chaired or served on several NRC committees, including the Committee on Design and Evaluation of Safer Chemical Substitutions: A Framework to Inform Government and Industry Decisions, the Committee to Review EPA's Draft IRIS Assessment of Formaldehyde, and the Committee to Review the IRIS Process. He has served on other advisory boards for the US Navy, NASA, and USDA, and is currently a member of NTP's Board of Scientific Counselors. Dr. Dorman is an elected fellow of the Academy of Toxicological Sciences and a fellow of the American Association for the Advancement of Sciences. He received a DVM from Colorado State University. He completed a combined PhD and residency program in toxicology at the University of Illinois at Urbana-Champaign, and he is a diplomate of the American Board of Veterinary Toxicology and the American Board of Toxicology.
• Andrew A. Rooney is deputy director of the Office of Health Assessment and Translation (OHAT) in the National Toxicology Program at the National Institute of Environmental Health Sciences. He has been developing risk assessment methods and guidance throughout his professional career and is a principal author of the 2012 WHO/IPCS Guidance for Immunotoxicity Risk Assessment for Chemicals. Most recently, he has been working on emerging issues in toxicology and environmental health, including methods to address study quality in terms of risk of bias for human, animal, and mechanistic studies and adaptation of systematic review methods for addressing environmental health questions. He led the team that developed the OHAT approach to systematic review. Dr. Rooney has an MS and a PhD in zoology from the University of Florida.

BDE-47

Studies of BDE-47 and effects on learning (see Table E4-1) and memory (see Table E4-2) were available.

Learning: There is moderate confidence in the body of evidence on developmental exposure to BDE-47 and effects on learning in rodents. Six studies in mice and rats were available. The two studies in rats found several indications of decreased learning in the Morris water maze (e.g., prolonged latency periods) after treatment with BDE-47 at doses of 1, 5, or 10 mg/kg-day on PND 10. Both studies were from the same laboratory (He et al. 2009, 2011). Three of the four mouse studies reported decreased learning in at least one test, strain, or sex and were conducted by different research groups (Eriksson et al. 2001; Ta et al. 2011; Koenigs et al. 2012; Woods et al. 2012). Nevertheless, the mouse results were variable across all the tests administered and a clear pattern was not identified to explain the heterogeneity in response relative to a susceptible strain, sex, or dose.

• Risk of bias: Downgraded because all studies had at least one rating of probably high or definitely high risk of bias in one of the key issues (e.g., lack of randomization of treatment), and most of the studies had multiple risk of bias issues, including not controlling for litter effects in the study design or analysis (see Figure E4-1).
• Unexplained inconsistencies: A qualitative evaluation of the evidence suggested a possible downgrade because of the heterogeneity in the evidence. However, a meta-analysis of studies of several PBDEs (see Chapter 4 and Appendix E, Section E-5), including BDE-47, and latency in the last trial of the Morris water maze showed consistent evidence of an effect on this measure of learning, so confidence was not downgraded.
• Indirectness: No downgrade because tests used are considered direct measures of learning.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade. Only two studies in rats were available, and the studies were from the same laboratory. The evidence base was judged to be inadequate for making judgments about cross-species consistency.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

TABLE E4-1

Studies of BDE-47 and Learning in Rodents.

FIGURE E4-1

Risk of bias heatmap of studies of BDE-47 and learning in rodents. In HAWC: https://hawcproject.org/summary/visual/353/.

Memory: There is low confidence in the body of evidence on developmental exposure to BDE-47 and effects on memory in rodents. Five studies in rats and mice were available. The one study in rats (He et al. 2011) reported decreased memory in the Morris water maze (e.g., prolonged latency periods) after exposure at 1, 5, and 10 mg/kg-day on PND 10. Three of the mouse studies (Eriksson et al. 2001; Ta et al. 2011; Koenig et al. 2012) reported no effects on memory at doses up to 12 mg/kg-day; however, Woods et al. (2012) reported decrements in memory in female Mecp2 308+/− mice with no effects on males or in C57BL6 mice of either sex. The body of evidence contained only a single study in rats and inconsistent results in mice. The dataset is similar and contains some of the same studies discussed above with respect to effects of BDE-47 on learning; fewer studies reported an effect, however, and one less study overall.

• Risk of bias: Downgraded because all the studies had a probably high risk of bias rating for at least one major issue (e.g., researchers were not blinded to the study groups during outcome assessment), and most of the studies had multiple risk of bias issues, including not controlling for litter effects in the study design or analysis (Eriksson et al. 2001; Woods et al. 2012) (see Figure E4-2).
• Unexplained inconsistencies: Downgraded because of the heterogeneity of the evidence.
• Indirectness: No downgrade because tests used are considered direct measures of memory.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade. Only one study in rats was available, so the evidence base was judged to be inadequate to make judgments about cross-species consistency.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

TABLE E4-2

Studies of BDE-47 and Memory in Rodents.

FIGURE E4-2

Risk of bias heatmap of studies of BDE-47 and memory in rodents. In HAWC: https://hawcproject.org/summary/visual/354/.

BDE-99

Studies of BDE-99 and effects on learning (see Table E4-3) and memory (see Table E4-4) were available.

TABLE E4-3

Studies of BDE-99 and Learning in Rodents.

Learning: There is moderate confidence in the body of evidence on developmental exposure to BDE-99 and learning in mice and rats based on five studies. Two of the three studies in rats reported longer latency during the acquisition period of tests using the Morris water maze at a dose of 2 mg/kg-day (Cheng et al. 2009; Blanco et al. 2013). Zhao et al. (2014) reported no effects at a lower dose (0.2 mg/kg-day) under similar exposure and testing conditions. In contrast, developmental exposure of Wistar rats at doses up to 30 mg/kg-day had no effect on learning tested with a Y maze (Llansola et al. 2009). A single study (Fischer et al. 2008) in NMRI mice also reported decrements in learning during the acquisition period in tests using either a radial maze or a Morris water maze at a dose of 0.8 mg/kg-day.

• Risk of bias: Downgraded because of serious concerns about several risk of bias issues. All of the studies were rated as having probably high risk of bias for at least one key risk of bias issue (e.g., researchers were not blinded to the study groups during outcome assessment), two of the studies had a definitely high risk of bias rating for not controlling for litter effects in the study design or analysis, and most of the studies had multiple risk of bias issues (see Figure E4-3).
• Unexplained inconsistencies: A qualitative evaluation of the evidence suggested a possible downgrade because of the heterogeneity in the evidence. Nevertheless, a meta-analysis of studies of several PBDEs (see Chapter 4 and Appendix E, Section E-5), including BDE-99, and latency in the last trial of the Morris water maze showed consistent evidence of an effect on this measure of learning, so confidence was not downgraded.
• Indirectness: No downgrade because tests used were considered direct measures of learning.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

FIGURE E4-3

Risk of bias heatmap of studies of BDE-99 and learning in rodents. In HAWC: https://hawcproject.org/summary/visual/355/.

TABLE E4-4

Studies of BDE-99 and Memory in Rodents.

Memory: There is moderate confidence in the data to evaluate whether developmental exposure to BDE-99 affects memory in rodents. The three available studies found no effects in several memory tests at doses of 0.2-2 mg/kg-day.

• Risk of bias: Downgraded because of serious concerns about several risk of bias issues. All the studies had a rating of probably high risk of bias for at least one key risk of bias issue (e.g., researchers were not blinded to the study groups during outcome assessment) and one study had a definitely high risk of bias rating for not controlling for litter effects in the study design or analysis (see Figure E4-4).
• Unexplained inconsistencies: No downgrade for inconsistency.
• Indirectness: No downgrade because tests used are considered direct measures of memory.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade because one study in mice and two in rats is insufficient to upgrade for evidence of consistency across species for a no-effect finding.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

FIGURE E4-4

Risk of bias heatmap of studies of BDE-99 and memory in rodents. In HAWC: https://hawcproject.org/summary/visual/356/.

BDE-153

Studies of BDE-153 and effects on learning (see Table E4-5) and memory (see Table E4-6) were available.

TABLE E4-5

Studies of BDE-153 and Learning in Rodents.

Learning: There is low confidence in the body of evidence to evaluate whether developmental exposure to BDE-153 affects learning in mice or rats. Two studies were available, one in mice and one in rats. Viberg et al. (2003) reported longer latencies in the acquisition period in the Morris water maze when mice were exposed to BDE-153 at 0.9 or 9 mg/kg-day on PND 10. The rat study (Zhang et al. 2013) reported no effect of BDE-153 at 10 mg/kg-day in performance in either the Morris water maze or the passive avoidance test when evaluated at PND 40 or PND 70.

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including lack of randomization of treatment; reduced confidence in outcome assessment due to lack of blinding of outcome assessors; and definitely high risk of bias ratings for not controlling for litter effects in the study design or analysis (see Figure E4-5).
• Unexplained inconsistencies: A qualitative evaluation of the evidence suggested a possible downgrade because of the heterogeneity in the evidence. Nevertheless, a meta-analysis of studies of several PBDEs (see Chapter 4 and Appendix E, Section E-5), including BDE-153, and of latency in the last trial of the Morris water maze showed consistent evidence of an effect on this measure of learning, so confidence was not downgraded.
• Indirectness: No downgrade because the tests used are considered direct measures of learning.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade because no evidence of consistency across species.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

FIGURE E4-5

Risk of bias heatmap of studies of BDE-153 and learning or memory in rodents. In HAWC: https://hawcproject.org/summary/visual/357/.

TABLE E4-6

Studies of BDE-153 and Memory in Rodents.

Memory: There is low confidence in the body of evidence on developmental exposure to BDE-153 and effect on memory in rodents. One study in mice and one in rats reported effects. (The two studies are the same ones that tested the effects of BDE-153 on learning.) The mouse study (Viberg et al. 2003) reported longer latencies in the relearning period of mice tested at 6 months of age in the Morris water maze after exposure to BDE-153 at 0.9 or 9 mg/kg-day. The rat study (Zhang et al. 2013) reported increased swimming time in the Morris water maze 1 month after treatment with BDE-153 at 5 and 10 mg/kg-day evaluated at PND 40 or PND 70 and memory impairment in the passive avoidance test at 10 mg/kg-day at PND 70. The two studies report results that were consistent in direction (both decreased performance on a memory test) across species.

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including lack of randomization of treatment; reduced confidence in outcome assessment due to lack of blinding of outcome assessors; and definitely high risk of bias ratings for not controlling for litter effects in the study design or analysis. (See heatmap in Figure E4-5.)
• Unexplained inconsistencies: Confidence is usually downgraded if only one study in each tested species is available because the database is insufficient to establish or evaluate consistency for a particular species. Consistency across species (see below), however, would be a reason to upgrade confidence. Considering both factors, there was no downgrade for unexplained inconsistency.
• Indirectness: No upgrade because tests were considered direct measures of memory.
• Imprecision: No upgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No change, no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: Confidence is usually upgraded if consistent results are observed across species. As noted above, however, it was not possible to evaluate consistency for each species because of the small data set. Considering both factors, there was no upgrade for consistency.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

BDE-203

One study of BDE-203 and effects on learning (see Table E4-7) or memory (see Table E4-8) was found.

TABLE E4-7

Studies of BDE-203 and Learning in Mice.

Learning: There is very low confidence in the data to evaluate whether developmental exposure to BDE-203 affects learning in mice from the single study available. The results suggest that timing of exposure might have an influence on effects because exposure to BDE-203 at 16.8 mg/kg-day on PND 10 affected learning during the acquisition period, whereas exposure on PND 3 had no effect on learning (Viberg et al. 2006).

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including lack of randomization of treatment; reduced confidence in outcome assessment due to lack of blinding of outcome assessors and a definitely high risk of bias rating for not controlling for litter effects in the study design or analysis (see Figure E4-6).
• Unexplained inconsistencies: Downgraded because unable to establish or evaluate consistency because the study did not have elements that would strengthen conclusions from a single study, such as multiple species, strains, or particularly large sample sizes (n = 50-100).
• Indirectness: No downgrade because test used is considered a direct measure of learning.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because only one dose was tested.
• Cross-species consistency: No upgrade because only mice were tested.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

Memory: There is very low confidence in the data to evaluate whether developmental exposure to BDE-203 affects memory in mice because the single study found no effect at a single dose (16.8 mg/kg-day). The study is the same one that also assessed learning.

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including lack of randomization of treatment; reduced confidence in outcome assessment due to lack of blinding of outcome assessors; and a definitely high risk of bias rating for not controlling for litter effects in the study design or analysis. (See heatmap in Figure E4-6.)
• Unexplained inconsistencies: Downgraded because unable to establish or evaluate consistency because the study did not have elements that would strengthen conclusions from a single study, such as multiple species, strains, or particularly large sample sizes (n = 50-100).
• Indirectness: No downgrade because test used is considered a direct measure of memory.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because only one dose was tested.
• Cross-species consistency: No upgrade because only mice were tested.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

FIGURE E4-6

Risk of bias heatmap of study of BDE-203 and learning and memory and BDE-206 and learning in mice. In HAWC: https://hawcproject.org/summary/visual/358/.

TABLE E4-8

Studies of BDE-203 and Memory in Mice.

BDE-206

One study of BDE-206 and effects on learning was found (see Table E4-9).

Learning: There is very low confidence in the data to evaluate whether developmental exposure to BDE-206 affects learning in mice as the single study found no effect at a single exposure level (16.8 mg/kg-day). The study is the same one that also tested BDE-203 (see above).

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including lack of randomization of treatment; reduced confidence in outcome assessment due to lack of blinding of outcome assessors; and a definitely high risk of bias rating for not controlling for litter effects in the study design or analysis. (see heatmap in Figure E4-6.)
• Unexplained inconsistencies: Downgraded because unable to establish or evaluate consistency because the study did not have elements that would strengthen conclusions from a single study, such as multiple species, strains, or particularly large sample sizes (n = 50-100).
• Indirectness: No downgrade because test used is considered a direct measure of learning.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because only one dose was tested.
• Cross-species consistency: No upgrade because only mice were tested.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

TABLE E4-9

Studies of BDE-206 and Learning in Mice.

BDE-209

Studies of BDE-209 and effects on learning (see Table E4-10) and memory (see Table E4-11) were available.

Learning: There is moderate confidence in the body of evidence to evaluate whether developmental exposure to BDE-209 affects learning in rodents. Multiple studies show effects on learning at doses of 20 mg/kg-day or greater when learning was assessed using a Morris water maze; other studies, however, show no effects in the same dose range using other test methods.

• Risk of bias: Downgraded because of serious concerns about multiple risk of bias issues, including reduced confidence in outcome assessment due to lack of blinding of outcome assessors in all studies and a definitely high risk of bias rating in three studies because of failure to control for litter effects in the study design or analysis (see Figure E4-7).
• Unexplained inconsistencies: A qualitative evaluation of the evidence suggested a possible downgrade because of the heterogeneity in the evidence. Nevertheless, a meta-analysis of studies of several PBDEs (see Chapter 4 and Appendix E, Section E-5), including BDE-209, and of latency in the last trial of the Morris water maze showed consistent evidence of an effect on this measure of learning, so confidence was not downgraded.
• Indirectness: No downgrade because tests used were considered direct measures of learning.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade because of inconsistencies in the results between rat and mouse studies.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

TABLE E4-10

Studies of BDE-209 and Learning in Rodents.

FIGURE E4-7

Risk of bias heatmap of studies of BDE-209 and learning in rodents. In HAWC: https://hawcproject.org/summary/visual/349/.

Memory: There is low confidence in the body of evidence to evaluate whether developmental exposure to BDE-209 affects learning in rodents. Multiple mouse studies show effects on memory at doses of 3.4 mg/kg-day or greater when memory was assessed using a Morris water maze; other studies, however, show no effects on memory at the same dose range using other methods.

• Risk of bias: Downgraded because of serious concerns about multiple risk of bias issues, including reduced confidence in outcome assessment due to lack of blinding of outcome assessors in all studies and a definitely high risk of bias rating in three studies because of failure to control for litter effects in the study design or analysis (see Figure E4-8).
• Unexplained inconsistencies: Downgraded for inconsistency.
• Indirectness: No downgrade because tests used were considered direct measures of memory.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade because of inconsistencies in the results between rat and mouse studies.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

TABLE E4-11

Studies of BDE-209 and Memory in Rodents.

FIGURE E4-8

Risk of bias heatmap of studies of BDE-209 and memory in rodents. In HAWC: https://hawcproject.org/summary/visual/350/.

DE-71

Studies of DE-71 and effects on learning (see Table E4-12), memory (see Table E4-13), and attention (see Table E4-14) were available.

Learning: There is very low confidence in the body of evidence on developmental exposure to DE-71 and effects on learning in rats. The results of the three available studies were inconsistent and used different tests (Morris water maze, radial maze, and visual discrimination) and animals of different ages. One study (Dufault et al. 2005) reported increased errors in the visual discrimination task at the single dose tested (30 mg/kg-day). The other two studies reported no effects of DE-71 on learning at the same dose using longer exposure windows; however, the animals in these studies were evaluated at older ages than were the rats tested in the Dufault et al. (2005) study.

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including reduced confidence in outcome assessment due to lack of blinding of outcome assessors and a definitely high risk of bias rating for exposure characterization in two of the studies (see Figure E4-9).

TABLE E4-12

Studies of DE-71 and Learning in Rats.

FIGURE E4-9

Risk of bias heatmap of studies of DE-71 and learning in rats. In HAWC: https://hawcproject.org/summary/visual/344/.

TABLE E4-13

Studies of DE-71 and Memory in Rats.

Memory: There is very low confidence in the body of evidence on developmental exposure to DE-71 and effects on memory in rats. The results of the two available studies were inconsistent and were evaluated in animals of different ages and with different tests (Morris water maze and radial maze). One study (de-Miranda et al. 2016) reported a reference memory deficit in female Wistar rats (not males) in the radial maze at the single dose tested (30 mg/kg-day).

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including reduced confidence in outcome assessment due to lack of blinding of outcome assessors and a definitely high risk of bias rating for exposure characterization in one of the studies (see Figure E4-10).
• Unexplained inconsistencies: Downgrade for inconsistency.
• Indirectness: No downgrade because tests used are considered direct measures of memory.
• Imprecision: No downgrade because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade because only rats were tested.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

FIGURE E4-10

Risk of bias heatmap of studies of DE-71 and memory in rats. In HAWC: https://hawcproject.org/summary/visual/345/.

TABLE E4-14

Studies of DE-71 and Attention in Rats.

Attention: There is very low confidence in the body of evidence on developmental exposure to DE-71 and effects on attention in rats. All of the data are from a single laboratory (Dufault et al. 2005; Driscoll et al. 2009, 2012) and the majority of the tests reported no effects at doses up to 30 mg/kg-day across multiple tests (various attention tasks and a visual task). In one experiment (Driscoll et al. 2009), rats exposed to DE-71 at 4.5 mg/kg-day demonstrated lower accuracy in Attention Task 1.

• Risk of bias: Downgraded twice because of serious concerns about multiple risk of bias issues, including reduced confidence in outcome assessment due to lack of blinding of outcome assessors and a definitely high risk of bias rating for exposure characterization in one of the studies (see Figure E4-11).
• Unexplained inconsistencies: Downgrade for inconsistency.
• Indirectness: No downgrade because tests used are considered direct measures of attention.
• Imprecision: No downgraded because no or minimal indications of large standard deviations (i.e., SD > mean).
• Dose-response: No upgrade because no clear evidence of dose-response gradient within or across studies.
• Cross-species consistency: No upgrade because only rats were tested.
• Other potential downgrades or upgrades: No evidence of publication bias (see Section E-3), large magnitude of effect, or residual confounding or other related factors that would affect confidence in the estimated effect.

FIGURE E4-11

Risk of bias heatmap of studies of DE-71 and attention in rats. In HAWC: https://hawcproject.org/summary/visual/347/.

Meta-Analyses on Combined Data on PBDEs

TABLE E5-1

Overall Analyses and Sensitivity Analyses of Studies of PBDEs and Latency in Last Trial of the Morris Water Maze.

FIGURE E5-1Risk of bias heatmap of studies of PBDEs and latency in last trial of the Morris water maze with standard deviations reported or digitized from figures in the publication

In HAWC: https://hawcproject.org/summary/visual/364/.

FIGURE E5-2Risk of bias heatmap of studies of PBDEs and latency in last trial of the Morris water maze without standard deviations

In HAWC: https://hawcproject.org/summary/visual/365/.

FIGURE E5-3

Benchmark dose estimates from studies of PBDEs and latency in last trial of the Morris water maze in rats and mice. Points without error bars are studies for which a standard deviation was not reported or could not be digitized from figures in the publication. (more...)

Meta-Analyses on Individual PBDEs

BDE-47

• Statistically significant overall effect. Heterogeneity (I² = 44%), but it was not statistically significant. Overall effect was robust to using only highest dose from each study.
• Positive trends in log₁₀(dose) and in dose, but only the latter was statistically significant. Reduced heterogeneity for log₁₀(dose) and linear model. Benchmark dose for a 5% change was estimated to be 1.4 mg/kg-day (95% CI: 1.0, 2.4) from the linear model and 0.83 mg/kg-day (95% CI: 0.34, 5.9) from the linear-quadratic model.

FIGURE E5-4Results of meta-analysis of studies of BDE-47 and latency in last trial of the Morris water maze

TABLE E5-2Overall Analyses and Sensitivity Analyses of Studies BDE-47 and Latency in Last Trial of the Morris Water Maze

View in own window

Analysis	Estimate	Beta	CI, Lower Bound	CI, Upper Bound	P value	tau	I2	P value for Heterogeneity	AICc
Primary Analyses
Overall	intrcpt	23.56	14.04	33.07	0.0000	6.72	44.43	0.1092	49.01*
Trend in log10(dose)	log10(dose)	7.19	-6.23	20.61	0.2939	4.72	27.79	0.1610	54.64
Linear in dose10	dose10	34.55	20.13	48.97	0.0000	6.76	41.56	0.0957	50.90
Linear-Quadratic in dose10	dose10	60.96	-27.12	149.04	0.1750	10.24	48.02	0.0829	58.02
Linear-Quadratic in dose10	I(dose10^2)	-29.19	-124.65	66.27	0.5490	10.24	48.02	0.0829	58.02
Sensitivity Analyses
Highest Doses-Overall	intrcpt	31.87	23.15	40.59	0.0000	0.00	0.00	0.2638	34.52

*

Indicates the lowest AICc.

FIGURE E5-5Benchmark dose estimates from studies of BDE-47 and latency in last trial of the Morris water maze

BDE-153

• Statistically significant overall effect; no heterogeneity. Too few data for a sensitivity analysis.
• Positive trend, but not a statistically significant trend. Only central estimate and lower bound could be estimated for a benchmark dose for a 5% change: 1.2 mg/kg-day (95% CI: 0.6, >10).

TABLE E5-3Overall Analyses and Sensitivity Analyses of Studies BDE-153 and Latency in Last Trial of the Morris Water Maze

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Analysis	Estimate	Beta	CI, Lower Bound	CI, Upper Bound	P value	tau	I2	P value for Heterogeneity	AICc
Primary Analyses
Overall	intrcpt	25.40	-0.18	50.99	0.052	0	0	0.82	32.56*
Trend in log10(dose)	log10(dose)	14.03	-30.94	58.99	0.541	0	0	0.88	38.16
Linear in dose10	dose10	41.17	-4.69	87.04	0.078	0	0	0.58	33.38
Linear-Quadratic in dose10	dose10	304.47	-193.63	802.57	0.231	0	0	0.93	38.17
Linear-Quadratic in dose10	I(dose10^2)	-295.30	-851.58	260.97	0.298	0	0	0.93	38.17

*

Indicates the lowest AICc.

FIGURE E5-6Results of meta-analysis of studies of BDE-153 and latency in last trial of the Morris water maze

BDE-209

• Statistically significant overall effect. Heterogeneity (I² = 42%), but it was not statistically significant. Overall effect was robust to using only highest dose from each study (only two studies).
• Statistically significant trends in log₁₀(dose) and linear trend in dose, with reduced heterogeneity. BMD estimates for a 5% change were 6.3 mg/kg-day (95% CI: 4.8, 9.2) from the linear model and 3.5 mg/kg-day (95% CI: 2.2, 7.9) from the linear-quadratic model.

FIGURE E5-7Benchmark dose estimates from studies of BDE-153 and latency in last trial of the Morris water maze in rats and mice

TABLE E5-4Overall Analyses and Sensitivity Analyses of Studies BDE-209 and Latency in Last Trial of the Morris Water Maze

View in own window

Analysis	Estimate	Beta	CI, Lower Bound	CI, Upper Bound	P value	tau	I2	P value for Heterogeneity	AICc
Primary Analyses
Overall	intrcpt	26.69	16.79	36.60	0.00000	6.36	42.27	0.12	41.15*
Trend in log10(dose)	log10(dose)	23.92	0.01	47.83	0.04990	0.00	0.00	0.37	46.48
Linear in dose10	dose10	7.70	5.32	10.08	0.00000	4.01	22.14	0.16	41.33
Linear-Quadratic in dose10	dose10	14.68	5.98	23.39	0.00094	0.00	0.00	0.27	46.94
Linear-Quadratic in dose10	I(dose10^2)	-1.60	-3.53	0.33	0.10377	0.00	0.00	0.27	46.94
Sensitivity Analyses
Highest Doses-Overall	intrcpt	39.61	9.96	69.26	0.00883	14.90	18.95	0.27	25.90

*

Indicates the lowest AICc.

FIGURE E5-8Results of meta-analysis of studies of BDE-209 and latency in last trial of the Morris water maze

FIGURE E5-9Benchmark dose estimates from studies of BDE-209 and latency in last trial of the Morris water maze

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