Reference	Comments
Li et al. (2017b). Hexabromocyclododecane‐induced Genotoxicity in Cultured Human Breast Cells through DNA Damage.	Largely observational study. HBL‐100 cells were exposed to high concentrations (0, 5, 10 and 50 mg/L) for 24 h. LDH leakage observed at 5 mg/L and significant ROS and comet tail migration at 50 mg/L. Expression of transcripts for ATM, and DNA repair genes OGG1 and MTH was increased and that for tumour suppressor gene BRCA1 downregulated.
Krivoshiev et al. (2015). Elucidating toxicological mechanisms of current flame retardants using a bacterial gene profiling assay.	Unclear what concentration was used and genes < 2‐fold regulated by HBCDDs.
Wielogorska et al. (2015). Endocrine disruptor activity of multiple environmental food chain contaminants.	Mammalian reporter gene assay for ER activation by β‐HBCDD. No activation found.
Kovarich et al. (2011). Quantitative structure‐activity relationship classification models for prediction of endocrine disrupting activity of brominated flame retardants.	γ‐HBCDD predicted to be anti‐oestrogenic by QSAR.
Papa et al. (2013). QSAR prediction of the competitive interaction of emerging halogenated pollutants with human transthyretin SAR QSAR.	γ‐HBCDD used in training set as a BFR without T4‐TTR competing potency.
Anisuzzaman and Whalen (2016). Tetrabromobisphenol A and hexabromocyclododecane alter secretion of IL‐1β from human immune cells.	Human natural killer (NK) cells, monocyte‐depleted (MD) peripheral blood mononuclear cells (MD‐PBMC) and PBMC exposed to 0.05–5.0 mM of HBCDD for 24 h, 48 h and 6 days. Interfere with the ability of immune cells to secrete IL‐1B. Exposure to HBCDD from 0.5–5.0 mM caused increases in IL‐1B secretion. Inhibitors of ERK1/2 and Casp1 ameliorated HBCDD induced IL‐1B release.
Anisuzzaman and Whalen (2016). Hexabromocyclododecane and tetrabromobisphenol A alter secretion of interferon gamma (IFN‐gamma) from human immune cells.	Human natural killer (NK) cells, monocyte‐depleted (MD) peripheral blood mononuclear cells (MD‐PBMC) and PBMC exposed to 0.05–5.0 mM of HBCDD for 24 h, 48 h and 6 days. interfere with the ability of immune cells to secrete IFN‐gamma. HBCDD stimulated secretion if IFN gamma but response very different in cells from different donors. Also baseline values were different.
Cato et al. (2014). Brominated flame retardants, tetrabromobisphenol A and hexabromocyclododecane, activate mitogen‐activated protein kinases (MAPKs) in human natural killer cells.	HBCDDs interfere with NK‐cell(s) lytic function. HBCDDs at 2.5 uM and above increased phosphorylation of p44/42; 1 uM and above increase phospho‐MEK1/2. p44/42 is required for NK lysis of tumour target cells
Hinkson and Whalen (2010). Hexabromocyclododecane decreases tumor‐cell‐binding capacity and cell‐surface protein expression of human natural killer cells.	Exposure of NK‐cells to 10 μM HBCDDs for 24 h caused > 90% loss of lytic function of NK‐cells and 71% decrease in NK‐cell binding function, and in expression of cell surface markers CD16 (58%) and CD56 (25%). NK‐cells exposed to 10 μM HBCDDs for 1 h followed by 24 h in HBCDD‐free media showed 89% loss of lytic function and decreased binding function (79.2%), and CD 16 expression (48.1%).
Koike et al. (2016). Brominated flame retardants, hexabromocyclododecane and tetrabromobisphenol A, affect proinflammatory protein expression in human bronchial epithelial cells via disruption of intracellular signalling.	HBCDDs exposure (10 μg/mL) increased the expression of ICAM‐1 and the production of IL‐6 and ‐8 in human bronchial epithelial cells (BEAS‐2B). HBCDDs caused activation of nuclear factor‐kappa B (p50, p65) and activator protein 1 (c‐Jun). HBCDDs showed no binding to nuclear oestrogen or thyroid hormone receptors.
Steves et al. (2018). Ubiquitous Flame‐Retardant Toxicants Impair Spermatogenesis in a Human Stem Cell Model.	Human stem cell‐based model of spermatogenesis, indicating that HBCDDs affect spermatogonia and primary spermatocytes through mitochondrial membrane potential perturbation and ROS generation, resulting in apoptosis. HBCDD concentrations: 1 uM, 10 uM, 25 uM, 50 uM, 100 uM and 200 μM dissolved in dimethyl sulfoxide (DMSO). Reduced viability at 25 μM but still 80% viability at 200 μM. Loss of marker (PLZF) for stem and progenitor spermatogonia observed at 1 μM. Primary spermatocyte marker piwi like RNA‐mediated gene silencing 2 (HILI) increased in expression from 1 to 25 μM and then declined.
Yasmin and Whalen (2018). Flame retardants, hexabromocyclododecane (HCBD) and tetrabromobisphenol a (TBBPA), alter secretion of tumor necrosis factor alpha (TNFα) from human immune cells.	Effects of HBCDDs on secretion of tumour necrosis factor alpha (TNFα) from human immune cells. In presence of T‐cells, HBCDDs increased TNFα secretion, but without T‐cells HBCDD caused reduction. HBCDD‐induced increases in TNFα secretion utilised the p38 MARK pathway.
Zhang et al. (2015). Transcriptomic and metabolomic approaches to investigate the molecular responses of human cell lines exposed to the flame retardant hexabromocyclododecane (HBCD).	Transcriptomic and metabolomic effects of HBCDDs on A549 and HepG2/C3A cells. MTT assay for A549 and HepG2 cells indicated EC50 values of 27.4 μM and 63.0 μM, respectively. Little effect on gene expression or metabolome in either cell type up to the highest dose of 4 μM.
Zou et al. (2013). PI3K/Akt pathway mediates Nrf2/ARE activation in human L02 hepatocytes exposed to low‐concentration HBCDs.	Nanomolar concentrations of HBCDDs stimulate L02 cell proliferation in a DNA‐PKcs‐dependent manner, increase protein levels and nuclear translocation of Nrf2, leading to upregulation of its target gene HO‐1. The PI3K/Akt pathway is essential for HBCDD activation of the Nrf2‐ARE pathway L02 cells.
Dorosh et al. (2010). Assessing oestrogenic effects of brominated flame retardants hexabromocyclododecane and tetrabromobisphenol A on MCF‐7 cells.	E‐screen assay in MCF‐7 cells. HBCDDs increased cell proliferation and dose‐dependently increased gene expression of TFF1 with effects starting at 20–200 nM. Blocked by the anti‐oestrogen, ICI 182,780.
Arini et al. (2017). A cell‐free testing platform to screen chemicals of potential neurotoxic concern across twenty vertebrate species.	High throughput cell‐free neurochemical assay based on receptors and enzymes. Not much specific information on HBCDDs.
An et al. (2014a). Hexabromocyclododecane and polychlorinated biphenyls increase resistance of hepatocellular carcinoma cells to cisplatin through the phosphatidylinositol 3‐kinase/protein kinase B pathway.	HBCDDs reduces sensitivity of HCC cells (HepG2, MHCC97H and MHCC97L) to cisplatin through modulation of the NF‐kB pathway activation and p53 and associated with the PI3K/Akt pathway activity.
Al‐Mousa and Michelangeli (2014). The sarcoplasmic‐endoplasmic reticulum Ca(2+)‐ATPase (SERCA) is the likely molecular target for the acute toxicity of the brominated flame retardant hexabromocyclododecane (HBCD).	Six BFRs assessed for cytotoxicity and potency to inhibit SERCA. Strong Direct correlation (r=0.94) between the potencies of inducing cell death and inhibiting SERCA. Ki of HBCDDs for SERCA was 2.7 uM. Mechanistic studies indicate that HBCDDs prevent ATP binding to SERCA.
Al‐Mousa and Michelangeli (2012). Some commonly used brominated flame retardants cause Ca2+‐ATPase inhibition, beta‐amyloid peptide release and apoptosis in SH‐SY5Y neuronal cells.	HBCDDs caused dose‐dependent increase in [Ca2+]i and inhibits the SERCA with an apparent Ki of 3.5 μM in SH‐SY5Y human neuroblastoma cells. Increase in ROS formation, cytochrome C release and apoptosis.
Bastos Sales et al. (2013). Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation.	No effect of 10 μM HBCDDs on DNA methylation and adipocyte differentiation.
Christen et al. (2010). Some flame retardants and the antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro.	Studied androgenic and antiandrogenic activity of BFRs and antimicrobials in vitro in MDA‐kb2 cells. HBCDDs very low activity but enhances androgenic activity.
Fa et al. (2013). Acute effects of hexabromocyclododecane on Leydig cell cyclic nucleotide signaling and steroidogenesis in vitro.	HBCDDs inhibited basal and hCG‐stimulated cAMP production, but elevated basal steroidogenesis. HBCDDs decrease in mitochondrial membrane potential in untreated and hCG‐treated cells.
Fa et al. (2015). HBCDD‐induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells.	HBCDDs caused a sustained reduction in ATP level. Accumulation of cAMP and androgen were also reduced. There was inhibition in the expression of genes for steroidogenic enzymes, luteinising hormone receptor, regulatory and transport proteins and a decrease in abundance of StAR. Enzymatic experiments indicated loss of activities of (CYP11A1) and 17β‐hydroxysteroid dehydrogenase (HSD17β).
Fa et al. (2014). Hexabromocyclododecane facilitates FSH activation of ERK1/2 and AKT through epidermal growth factor receptor in rat granulosa cells.	HBCDDs potentiate FSH‐stimulated phosphorylation of EGFR, ERK1/2 and AKT, indicating a direct effect on EGFR.
Huang et al. (2016b). In vitro study on the biotransformation and cytotoxicity of three hexabromocyclododecane diastereoisomers in liver cells.	Cytotoxicity of HBCDDs in L02 and HepG2 cells was β‐HBCDD > γ‐HBCDD > α‐HBCDD.
Ibhazehiebo et al. (2011a). 1, 2, 5, 6, 9, 10‐αHexabromocyclododecane (HBCD) impairs thyroid hormone‐induced dendrite arborisation of Purkinje cells and suppresses thyroid hormone receptor‐mediated transcription.	HBCDDs at 0.1 nM suppressed TR‐mediated transcription in a reporter gene assay, and suppressed TH‐induced dendrite arborisation of Purkinje cells in primary cerebellar culture derived from rat neonates.
Ibhazehiebo et al. (2011b). Brain‐derived neurotrophic factor (BDNF) ameliorates the suppression of thyroid hormone‐induced granule cell neurite extension by hexabromocyclododecane (HBCD).	HBCDDs at 0.1 nM suppressed TH‐induced neurite extension of granule cell aggregate in primary rat cerebellar granule cell aggregate cultures. BDNF ameliorated this effect in presence of T3. Results indicate that T3‐stimulated increase in BDNF may be involved in HBCDD‐induced impairment of TH‐mediated neuritogenesis of granule cells.
Kim et al., 2016. Influence of hexabromocyclododecane and 4‐nonylphenol on the regulation of cell growth, apoptosis and migration in prostatic cancer cells Toxicol In Vitro, 32: 240‐7.	HBCDDs may enhance progression of prostate cancer by modulating growth and migration of LNCaP prostate cells by acting on cell cycle and apoptosis.
Koike et al. (2013). Brominated flame retardants stimulate mouse immune cells in vitro. J Appl Toxicol, 33(12): 1451‐9.	HBCDDs increased expression of T‐cell receptor, MHC class II and CD86 as well as IL‐4 production in splenocytes. Authors suggested that HBCDDs may induce or enhance immune/allergic responses by increasing antigen presentation‐related molecule expression and IL‐4 production.
Krivoshiev et al. (2016). Assessing in‐vitro estrogenic effects of currently‐used flame retardants.	HBCDDs stimulated proliferation of MCF‐7 cells with an EC20 of 5.5 μM. HBCDDs also inhibited 17B‐oestradiol stimulated proliferation with an IC20 of 17.6 μM.
Park et al. (2012). Cell growth of BG‐1 ovarian cancer cells is promoted by di‐n‐butyl phthalate and hexabromocyclododecane via upregulation of the cyclin D and cyclin‐dependent kinase‐4 genes.	0.2 μM HBCDDs stimulated proliferation of BG‐2 ovarian cancer cells. HBCDDs upregulated mRNA for cyclin D and cyclin‐dependent kinase‐4 (cdk‐4), which are downstream target genes of ER.
Reffatto et al. (2018). Parallel in vivo and in vitro transcriptomics analysis reveals calcium and zinc signalling in the brain as sensitive targets of HBCD neurotoxicity.	Neurotoxic potential of HBCDDs was studied in mouse brain and, to separate direct effects from system responses (e.g. via hormones) two cell lines of mouse neuronal origin, NSC‐19 and N2A, as well as in primary hippocampal neuronal cultures. Transcriptome profiling of mouse brains and the two cell lines indicated that sex steroid regulation, and Ca2+ and Zn2+ homeostasis in glutamatergic neurons were preferentially affected. Follow‐up experiments on the two cell lines and isolated primary hippocampal neurons confirmed effects of HBCDDs on free [Zn2+], glutamate‐induced post‐synaptic [Ca2+] and zinc‐stimulated post‐synaptic [Ca2+] release, which was almost completely blocked by 1 uM HBCDDs. The increase in cytosolic free [Zn2+] could be partially blocked by an antioxidant, indicating ROS formation as a contributing factor.
Saegusa et al. (2012). Transient aberration of neuronal development in the hippocampal dentate gyrus after developmental exposure to brominated flame retardants in rats.	Female pregnant SD rats were fed a diet containing 0 (control), 100, 1,000 or 10,000 mg/L of HBCDDs from GD 10 to PND20. Effects on neuronal development was observed at 1,000 and 10,000 mg/L. Reelin+ and NeuN+ cells in interneurons of the dentate hilus increased temporarily, suggestive of aberrant neuronal migration. Effect on reelin+ cells was only seen at 1,000 mg/L (not at 10,000 mg/L). An increase in apoptotic bodies was observed at PND20 in the subgranular zone. At PND20 there was a small reduction in serum T3.
Suzuki et al. (2013). Similarities in the endocrine‐disrupting potencies of indoor dust and flame retardants by using human osteosarcoma (U2OS) cell‐based reporter gene assays.	CALUX reporter gene assays, based on U2OS cells were used to evaluate reporter gene assays used to evaluate activities of flame retardants on the human androgen receptor (AR), oestrogen receptor α (ERα), progesterone receptor (PR), glucocorticoid receptor (GR) and peroxisome proliferator‐activated receptor γ2 (PPARγ2). HBCDD showed relatively mild antagonistic effect on the AR and PR receptors.
Wilson et al. (2016). Do persistent organic pollutants interact with the stress response? Individual compounds, and their mixtures, interaction with the glucocorticoid receptor.	HBCDDs were not tested alone but in chemical mixture.
Wu et al. (2016b). Hexabromocyclododecane exposure induces cardiac hypertrophy and arrhythmia by inhibiting miR‐1 expression via up‐regulation of the homeobox gene Nkx2.5.	Zebrafish and H9C2 rat cardiomyocyte cells. Zebrafish embryos were exposed to 0, 2, 20 and 200 nM HBCDD in the water. Exposure to 20 and 200 nM resulted in cardiac hypertrophy and increased deposition of collagen. miR‐1 might mediate HBCDD induced cardiac hypertrophy and arrhythmia via its target genes Mef2a and Irx5. H9C2 rat cardiomyocyte cells exposed to HBCDDs showed reduced Ca2+‐ATPase activity and elevated Ca2+ in the cytosol.
Zhang et al. (2016). Gene expression and metabolic responses of HepG2/C3A cells exposed to flame retardants and dust extracts at concentrations relevant to indoor environmental exposures.	Multiomics approach used to interrogate effects of dust extract and BFR mixture (including HBCDDs) in HepG2 cells. Exposure of cells to dust extracts induced expression of several CYP biotransformation enzymes. Such effects were not observed in the mixture of BFRs and it was concluded that other components of the dust extract were likely causing the observed effects.
Zhong et al. (2015). HBCD and PCBs enhance the cell migration and invasion of HepG2 via the PI3 K/Akt pathway.	HepG2 cells exposed to HBCDDs were assessed for cell viability, apoptosis, cell migration and invasion using cell counting kit‐8 (CCK‐8) assay, flow cytometry, cell scratch assay, respectively. 10 nM HBCDDs stimulated migration and invasion of HepG2 cells, increased protein expression of MMP9 and suppressed E‐cadherin (CDH1) expression. HBCDD exposure increased stimulatory phosphorulation of protein kinase B and extracellular signal‐regulated kinase 32 (ERK), and expression of mammalian target of rapamycin (mTOR).
Ziemiliska et al. (2012). Acute cytotoxicity evoked by tetrabromobisphenol A in primary cultures of rat cerebellar granule cells outweighs the effects of polychlorinated biphenyls.	Mild effect of 25 μM HBCDDs on calcium uptake in CGCs.
Zimmer et al. (2011). In vitro steroidogenic effects of mixtures of persistent organic pollutants (POPs) extracted from burbot (Lota lota) caught in two Norwegian lakes.	HBCDDs were not tested alone but in chemical mixture.
An et al. (2013). The cytological effects of HBCDs on human hepatocyte L02 and the potential molecular mechanism.	High concentration of HBCDDs (> 20 μM) decreased survival of L02 hepatocytes, lower dose of HBCDDs (10−13–10−7 M) stimulated cell proliferation and up‐regulation of PCNA protein expression level.
An et al. (2016). The ‘adaptive responses’ of low concentrations of HBCD in L02 cells and the underlying molecular mechanisms.	L02 cells were able to acquire tolerance to a high concentration of α‐HBCDD through pre‐exposure to a lower dose. They provided evidence that the mechanism of this is through activation of the PI3K/Akt pathway, reduced phosphorylation of AMPK and increased phosphorylation of p38 MAPK.
An et al. (2014b). Oligomeric proanthocyanidins alleviate hexabromocyclododecane‐induced cytotoxicity in HepG2 cells through regulation on ROS formation and mitochondrial pathway.	This study examined effects of oligomeric proanthocyanidins (OPCs) on cytotoxicity induced by HBCDDs. Whilst interesting it is not considered relevant to the RA of HBCDDs.
Canbaz et al. (2016). Indoor pollutant hexabromocyclododecane enhances house dust mite‐induced activation of human monocyte‐derived dendritic cells.	Simultaneous exposure of monocyte dendritic cells to house mite dust allergens and HBCDDs, and HBCDDs increased the expression of HLA‐DR, co‐stimulatory molecule CD86 and pro‐inflammatory cytokine IL‐8 depending on the dose of HBCDDs (1–20 μM). An increase in IL‐8 was observed at 1 μM but other effects required higher concentrations (10–20 μM).
Wang et al. (2016a). New Insights into the Cytotoxic Mechanism of Hexabromocyclododecane from a Metabolomic Approach.	Detailed study on HepG2 cells, starting with NMR and following up with hypothesis driven experiments and analyses. HepG2 cells exposed to reagent‐grade HBCDD formula (purity: > 95%, Aladdin Industrial Corp.) at 0.05, 1 and 10 mg/L. HBCDD exposure resulted in amino acid metabolism, protein biosynthesis, fatty acid metabolism and phospholipid metabolism. Beta oxidation of long‐chain fatty acids was suppressed, ATP production reduced, Na/K‐ATPase inhibited and uptake of amino acids and glucose impaired. Reduced beta oxidation was accompanied by increase in free fatty acids and increased synthesis of phospholipids. The authors suggest that most effects may be a results of reduced ATP production and consequential loss of Na/K‐ATPase and Ca‐ATPase activities, leading to reduced glucose and amino acid uptake by cells.
Georgantzopoulou et al. (2014). P‐gp efflux pump inhibition potential of common environmental contaminants determined in vitro.	The calcein‐acetoxymethyl ester (calcein‐AM) assay in P‐glycoprotein–overexpressing Madin–Darby canine kidney cells (MDCKII–MDR1) was exploited to study the inhibition of P‐gp efflux pumps by HBCDDs (technical mixture) and other aquatic contaminants. Data suggested that HBCDDs may stimulate the P‐gp efflux pump.
Kamata et al. (2018). Agonistic effects of diverse xenobiotics on the constitutive androstane receptor as detected in a recombinant yeast‐cell assay	HBCDDs have a relatively low potential to activate CAR in a recombinant yeast assay.
Lille‐Langoey et al. (2015). Environmental contaminants activate human and polar bear (Ursus maritimus) pregnane X receptors (PXR, NR1I2) differently.	COS‐7 cells transfected with luciferase reporter gene construct was used to assess activation of polar bear and human PXR. A technical HBCDD mixture containing 81% γ‐HBCDD was tested. The HBCDD mixture had a moderate potency to activate PXR compared with other chemicals tested and maximum activation was 41% of that obtained by rifampicin exposure.
Montano et al. (2011). Effects of mixtures of persistent organic pollutants (POPs) derived from cod liver oil on H295R steroidogenesis.	HBCDDs were not tested alone but in chemical mixture.
van den Dungen et al. (2017). Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.	DNA methylation and gene expression were studied in response to exposure to different POPs during human adipocyte differentiation. HBCDDs did not affect lipid accumulation in adipocytes. 1 μM HBCDD induced ALP activity in adipocytes.
van den Dungen et al. (2015). Steroid hormone related effects of marine persistent organic pollutants in human H295R adrenocortical carcinoma cells.	Effects of various POPs and mixtures thereof were studied in H295R adrenocortical carcinoma cells in relation to endocrine effects. HBCDDs did not affect any steroid hormone levels. 100 μM HBCDDs induced expression of CYP19A1.
Kang et al. (2012). Induced growth of BG‐1 ovarian cancer cells by 17beta‐estradiol or various endocrine disrupting chemicals was reversed by resveratrol via downregulation of cell cycle progression.	Treatment of BG‐1 cells with HBCDDs resulted in an increase of cell growth.
Li et al. (2017c). Neuroprotection by Taurine on HBCD‐Induced Apoptosis in PC12.	Exposure of PC12 cells to 10 μM HBCDDs reduced protein expression of Bcl‐2, increased expression in Bax protein and activity of caspase‐3. Taurine protected against these effects.
Liu et al. (2017). Taurine Alleviate Hexabromocyclododecane‐Induced Cytotoxicity in PC12 Cells via Inhibiting Oxidative Stress.	Taurine protected against cytotoxicity induced by HBCDDs in PC12 cells through inhibition of oxidative stress.