Marcia Hardy

The toxicology of the three commercial polybrominated diphenyl oxide (ether) flame retardants

Three commercial polybrominated diphenyl oxide flame retardants (PBDPO, PBDE) are manufactured: decabromodiphenyl oxide (DBDPO), octabromodiphenyl oxide (OBDPO) and pentabromodiphenyl oxide (PeBDPO). The composition, production volumes, uses and toxicology of the three products differ. In 1999, DBDPO accounted for approximately 82% of the global PBDPO usage. DBDPO has been extensively tested. DBDPO was not acutely toxic, was not irritating to the skin or eye, and did not induce skin sensitization. No evidence of genotoxic effects was detected in the Ames Salmonella, chromosome aberration, mouse lymphoma, or sister chromatid exchange tests. No cytogenic changes were observed in the bone marrow of rats (parents and offspring) undergoing a one-generation reproduction test. DBDPO did not adversely affect development or reproduction in rats. DBDPOs no-adverse-effect-level (NOAEL) in repeated dose studies was > or = 1000 mg/kg body weight. No, equivocal, or some evidence of carcinogenicity, dependent on genus and sex, was found in mice and rats at 2.5% and 5% of the diet administered for 2 years. DBPDO was poorly absorbed from the gastrointestinal tract (< 0.3-2% oral dose), had a short half-life (< 24 h) compared to PCB 153 (only 2% of an oral dose eliminated by rats in 21 days), and was rapidly eliminated via the feces (> 99% in 72 h). In contrast, components of the PeBDPO product were well absorbed and slowly eliminated, OBDPOs effect level in a 90-day study was approximately 100 mg/kg, PeBDPOs no-effect-level (NOEL) in a 30-day study was 1 mg/kg, and OBDPO induced developmental toxicity in the rat. In aquatic species, neither DBDPO nor OBDPO were toxic to aquatic organisms or bioconcentrating. Components of the PeBDPO product bioconcentrated in fish but produced little evidence of adverse effects.

A comparison of the properties of the major commercial PBDPO/PBDE product to those of major PBB and PCB products.

Decabromodiphenyl oxide (DBDPO), a highly effective polybrominated diphenyl oxide (PBDPO) flame retardant (FR) used primarily in electrical and electronic equipment, is the second highest volume brominated flame retardant (BFR) and accounts for 82% of the PBDPO usage globally. The apparent similarities in chemical structure between the DBDPO, polychlorinated and polybrominated biphenyl (PCB, PBB) molecules have led to the presumption that these substances also share similar toxicological and environmental properties. However, DBDPOs physical/chemical properties, applications, environmental release, and toxicology differ substantially from the former PCB/PBB products. DBDPO is a heavier and larger molecule than components of the predominant PCB/PBB products used in the past, and the commercial DBDPO product has a lower water solubility and vapor pressure than the former PCB and PBB products. DBDPOs detection in the environment is generally in sediments near known point sources, and its primary use in thermoplastics limits its environmental release from end products. PBB environmental release has been primarily associated with one accident occurring in the US in 1973. The PCBs, used in applications with a high potential for environmental release, were detected in diverse locations around the world as early as in the 1970s. Current releases of PCB are considered related to an environmental cycling process of congeners previously released into the environment; however, DBDPOs physical/chemical properties do not indicate a similar potential. Extensive testing of the DBDPO commercial product has demonstrated that it is toxicologically and pharmacokinetically different from the predominant PCB and PBB products used in the past. Thus, although the chemical structures of DBDPO, PBB, and PCB appear similar, the properties of DBDPO are distinctly different.

Toxicology and human health assessment of decabromodiphenyl ether

Recent public concern has focused on potential reproductive and developmental effects from exposure to low levels of bisphenol A (BPA, CAS number 80-05-7). Two previous published reviews (Gray et al., 2004a; Goodman et al., 2006) conducted weight-of-evidence evaluations of in vivo reproductive/developmental toxicity from BPA exposure < or = 5 mg/kg-d based on studies published through February 2006. Here, an update of those analyses presents additional relevant studies that were published through July 25, 2008, and a weight-of-evidence analysis of the studies evaluated in all three reviews. As with the earlier literature, positive findings: (1) are countered by null findings in more numerous studies; (2) have not been replicated; (3) do not exhibit coherence and plausibility; (4) do not show consistency across species, doses, and time points; and/or (5) were from studies using non-oral exposure routes. Owing to the lack of first-pass metabolism, results from non-oral studies are of limited relevance to human exposure. Exposure levels in most of the low-dose oral and non-oral animal studies are generally much higher than those experienced by even the most exposed people in the general population. The weight of evidence does not support the hypothesis that low oral doses of BPA adversely affect human reproductive and developmental health.We evaluated the available pharmacokinetic data and human and animal toxicity data for 2,2′,3,3′,4,4′,5,5′,6,6′-decabromodiphenyl ether (BDE-209) (CASRN 1163-19-5) with the objective of deriving a reference dose (RfD) based on the best available science. The available studies for deriving an RfD were first screened using the Klimisch criteria and further evaluated using the United States Environmental Protection Agency’s general assessment factors for data quality and relevance (i.e., soundness, applicability and utility, clarity and completeness, uncertainty and variability, and evaluation and review). The chronic 2-year dietary feeding study conducted by the United States National Toxicology Program (, Technical Report Series No. 309) was selected for RfD derivation. Hepatocellular degeneration in male rats was chosen as the critical endpoint in the development of an RfD. For dose-response characterization, we applied benchmark-dose modeling to animal data and determined a point of departure (the 95% lower confidence limit for a 10% increase in hepatocellular degeneration) of 419 mg/kg-day for oral exposures. Based on the similar pharmacokinetic characteristics of BDE-209 across species, this value was converted to a human equivalence dose of 113 mg/kg-day by applying a dosimetric adjustment factor based on body weight scaling to the ¾ power. An oral RfD of 4 mg/kg-day was calculated by using a composite uncertainty factor of 30, which consisted of 10 for intraspecies uncertainty, 3 for interspecies uncertainty (i.e., 3 for toxicodynamics × 1 for toxicokinetics), and 1 for deficiencies with the database. We consider the RfD to be adequately protective of sensitive subpopulations, including women, their fetuses, children, and people with hepatocellular diseases.

Regulatory status and environmental properties of brominated flame retardants undergoing risk assessment in the EU: DBDPO, OBDPO, PeBDPO and HBCD

Brominated flame retardants (BFRs) are a structurally diverse group of compounds; their major point in common is not their chemical structure but rather that of their use as flame retardants. BFRs undergoing risk assessment in the EU under the existing chemicals regulation are the polybrominated diphenyl oxides (ethers; PBDPO), decabromodiphenyl oxide (DBDPO), octabromodiphenyl oxide (OBDPO) and pentabromodiphenyl oxide (PeBDPO), and the cyclic aliphatic, hexabromocyclododecane (HBCD). This paper will address the toxicology and environmental properties of these flame retardants as well as research and regulatory activities affecting them. DBDPO, OBDPO, PeBDPO, and the polybrominated biphenyls (PBB) were included in OECDs Risk Reduction Programme on Selected BFRs and are included in our Voluntary Industry Commitment (VIC) accepted at the OECD Joint Meeting in June 1995. Previous reviews on some or all of the BFRs under consideration include the WHO EHC Document on the PBDPO, the Binetti report on flame retardants used in upholstered furniture in the EU, the UK risk policy analyst report on the PBDPO, the Netherlands reassessment of the PBB and PBDPO, the OECD Monograph on Selected BFRs, the techno-economic study on emissions of BFRs in the EU. Research is presently underway to add to the data base of knowledge on these BFRs. All have high molecular weights ranging from 564 (PeBDPO) to 959 (DBDPO) and negligible vapor pressures and water solubilities. Studies on the water solubility of the PBDPO and HBCD have recently been concluded; their water solubilities are <0.1 ppb (DBDPO), <1 ppb (OBDPO), 13 ppb (PeBDPO) and 3.4 ppb (HBCD). Their vapor pressure is so low (DBDPO <10–7 mm Hg) that common methods of analysis are not applicable and a new method, the spinning rotor method, is being validated for use. Adsorption to soil/sediment is expected to be high. Extraction studies on DBDPO and HBCD establish that migration of these BFRs from polymers into water is negligible. The same negligible migration characteristics are also anticipated for OBDPO and PeDPO. Therefore, contamination of groundwater as a result of disposal of flame retarded plastics should not occur. The above physical/chemical properties of these BFRs minimize their potential to move into and in the environment irrespective of their lack of ready biodegradability. In addition, DBDPO, which has been extensively studied, has been found to have a short half life in rats, minimal absorption from the gastrointestinal tract, rapid elimination, and to lack bioaccumulation potential in fish. These properties, coupled with the minimal effects on mammalian species on repeated dosing of DBDPO and HBCD, and their lack of mutagenicity and skin sensitization indicate these brominated flame retardants can be used by society to provide needed protection from the hazard of fire.

A comparison of the fish bioconcentration factors for brominated flame retardants with their nonbrominated analogues

Flame retardants (FR) play a significant role in reducing the flammability of many consumer products. On a volume basis, approximately 25% of the FRs in use today utilize bromine as the active flame-retarding moiety. Their applications are those requiring high FR performance or in resins needing an FR that is active during the gas phase. Laboratory fish bioconcentration factors (BCFs) for 11 brominated FRs (BFRs) or their components were compared with those for their nonbrominated analogues. Bioconcentration, defined here as a BCF of greater than 1,000, was not observed in those brominated molecules examined with molecular weights of 700 or greater. These included the decabromodiphenyl oxide and octabromodiphenyl oxide commercial products, ethane 1,2-bis(pentabromophenyl), ethylene bis-tetrabromophthalimide, and decabromobiphenyl. Tetrabromobisphenol A, with a molecular weight of less than 700, also did not bioconcentrate. This likely relates to the ease with which it is metabolized and eliminated. Within the BFR class of polybrominated diphenyl oxides/ethers, the BCFs for those congeners with molecular weights of between approximately 450 and 700 varied with the number and position of the bromine atoms. The BCF of hexabromocyclododecane appeared to be related to its cyclododecane substructure, not to its bromine content. Bioconcentration was not a characteristic feature of the BFRs examined here.

An oral developmental neurotoxicity study of decabromodiphenyl ether (DecaBDE) in rats

BACKGROUND Decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) is a flame retardant used in a variety of manufactured products. A single oral dose of 20.1 mg/kg administered to mice on postnatal day 3 has been reported to alter motor activity at 2, 4, and 6 months of age. METHODS To further evaluate these results, a developmental neurotoxicity study was conducted in the most commonly used species for studies of this type, the rat, according to international validated testing guidelines and Good Laboratory Practice Standards. DecaBDE was administered orally via gavage in corn oil to dams from gestation day 6 to weaning at doses of 0, 1, 10, 100, or 1,000 mg/kg/day. Standard measures of growth, development, and neurological endpoints were evaluated in the offspring. Motor activity was assessed at 2 months of age. Additional motor activity assessments were conducted at 4 and 6 months of age. Neuropathology and morphometry evaluations of the offspring were performed at weaning and adulthood. RESULTS No treatment-related neurobehavioral changes were observed in detailed clinical observations, startle response, or learning and memory tests. No test substance-related changes were noted in motor activity assessments performed at 2, 4, or 6 months of age. Finally, no treatment-related neuropathological or morphometric alterations were found. CONCLUSIONS Under the conditions of this study, the no-observed-adverse-effect level for developmental neurotoxicity of DecaBDE was 1,000 mg/kg/day, the highest dose tested.

Prenatal developmental toxicity of decabromodiphenyl ethane in the rat and rabbit.

BACKGROUND The potential embryotoxic and teratogenic effects of decabromodiphenyl ethane (DBDPEthane; CASRN 84852-53-9) were evaluated in prenatal developmental studies using rats and rabbits and performed in accordance with international guidelines and Good Laboratory Practice standards. Preliminary dose-range-finding studies were conducted, which indicated doses up to 1,250 mg/kg-day were well tolerated by both rats and rabbits. METHODS For the developmental studies, animals were administered DBDPEthane via gavage at dosage levels of 0, 125, 400, or 1,250 mg/kg-day from gestation day (GD) 6 through 15 for rats and GDs 6 through 18 for rabbits. All female rats and rabbits were sacrificed on GD 20 or GD 29, respectively, and subjected to cesarean section. Fetuses were individually weighed, sexed, and examined for external, visceral and skeletal abnormalities. RESULTS No treatment-related mortality, abortions, or clinical signs of toxicity were observed during the study. Body weights, body weight gain, and food consumption were not affected by treatment. No significant internal abnormalities were observed in either species on necropsy. Cesarean section parameters were comparable between control and treated groups. No treatment-induced malformations or developmental variations occurred. CONCLUSIONS Based on these results, no evidence of maternal toxicity, developmental toxicity, or teratogenicity was observed in rats or rabbits treated with DBDPEthane at dosage levels up to 1,250 mg/kg-day.

Effects of Dose, Administration Route, and/or Vehicle on Decabromodiphenyl Ether Concentrations in Plasma of Maternal, Fetal, and Neonatal Rats and in Milk of Maternal Rats

The effects of route and vehicle on blood and milk levels of decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) were investigated in the rat to assist in the design and conduct of a developmental neurotoxicity study. Blood plasma and/or milk concentrations were determined in dams, fetuses, and/or nursing pups after repeated DecaBDE administration by gavage throughout gestation or gestation and lactation using corn oil (CO) or soyaphospholipon/Lutrol F 127-water (SPL) as the vehicle. The impact of vehicle on plasma levels was also investigated in pups derived from naive dams after a single postnatal dose. This study reports for the first time fetal and neonatal plasma concentrations concurrent with those of maternal plasma and/or milk. Higher concentrations of DecaBDE were achieved in plasma and in milk with CO than with SPL. Furthermore, pups derived from dams treated with only SPL were lower in body weight, compared with those from dams treated with either CO, CO and DecaBDE, or SPL and DecaBDE. The study further shows that exposure to DecaBDE is relatively consistent across the dose range of 100 to 1000 mg/(kg · day) when administered in CO.

Use of the pup as the statistical unit in developmental neurotoxicity studies: overlooked model or poor research design?

Fischer et al. (2008) report that BDE-99 (2,2#4,4#,5pentabromodiphenyl ether) and methylmercury exposure to mouse pups on postnatal day 10 disrupted spontaneous behavior, reduced habitation, and impaired learning/memory abilities. The authors employed an experimental design developed in their laboratory stating: ‘‘[i]n this neonatal animal model each of the different treatment groups comprise mice from three to four different litters. Randomly selecting animals from at least three different litters will have the same statistical effect and power compared to the use of litter based studies to evaluate developmental neurotoxicity (DNT) in neonatal mice (Eriksson and Viberg, 2005; Eriksson et al., 2005).’’ This approach contradicts accepted practice in DNT studies. The experimental unit used for the statistical analysis of data derived from DNT studies is the litter, not the individual pup (EPA, 2000 [‘‘In general, data from developmental toxicity studies in rodents are best modeled using nested models. These models account for any intralitter correlation, or the tendency of littermates to respond similarly to one another relative to the other litters in a dose group,’’ at p. 73.]; OECD, 2007 [‘‘The statistical unit of measure should be the litter (or dam) and not the pup,’’ at p. 4.]). Litter effects over as few as three litters are generally large and statistically meaningful, and treating as few as two pups per litter as independent measurements, as done by Fischer et al., can almost triple the nominal 0.05 alpha level (Holson and Pearce, 1992). By way of support for their claim, Fischer et al. cite a book chapter (Eriksson and Viberg, 2005) and an abstract presented at the 44th Annual Meeting of the Society of Toxicology (SOT) (Eriksson et al., 2005). The book chapter provides a cursory description of the SOT abstract, and no data is provided in either. With respect to the abstract’s conclusion, Holson et al. (2007) said in their review of statistical issues and techniques for DNT testing that ‘‘[t]his conclusion is inaccurate,’’ ‘‘[t]here are litter effects on spontaneous motor activity,’’ and ‘‘[i]gnoring litter effects in the statistical analysis of DNT studies is simply not an acceptable practice’’. The Swedish research group has published eight papers since 2002 in Toxicological Sciences using the same design. This is troubling, because repeated publications tend to lend credence to this methodology. The impact of these papers, and others published elsewhere by this group using the same design, is not inconsequential. Their conclusions have resulted in the requirement that a formal guideline DNT test be performed for the European Union (ECB, 2002). Further, the results are driving legislation at the state level in the United States (MSL, 2004), and, at the federal level, where they have been used to set draft reference doses (IRIS, 2006a, b). Though not the primary topic of this letter, we also point out that the eight papers in Toxicological Sciences used a device called the ‘‘Rat-O-Matic’’ (ADEA Elektronik AB, Uppsala, Sweden) to measure the primary end point, for example, motor activity. In all eight papers, the supporting documentation given for the device was a dissertation (Fredriksson, 1994). We were unable to find peer-reviewed literature validating this piece of equipment’s use in rats or mice, nor were we able to find any further information on the device or its manufacturer other than a listing for the company on the World Trade Markets web site. Toxicological Sciences is not responsible for how its published research is used. However, Toxicological Sciences does have a professional responsibility to ensure that the peer review process used acts as a quality control system (Grainger, 2007). It is unfortunate that these eight papers were peerreviewed and subsequently published without adequate data supporting the validity of their design or measuring device, especially when the design obviates an accepted norm in developmental neurotoxicology studies.

Provisional human health risk assessment of PBDEs in sewage sludge used for agricultural purposes

Clarke et al. (2008) reported concentrations of polybrominated diphenyl ethers (PBDEs), as well as a hexabromobiphenyl, in sewage sludge from Australian wastewater treatment plants. The authors also provided a concise overview of reported levels of PBDEs in sewage sludge from around the world. Clarke et al. (2008) concluded that their work warrants a risk assessment of PBDEs in sewage sludge when used for land applications. Herein, we provide a provisional risk assessment by applying the paradigm outlined by the United States (US) National Academies’ National Research Council (NRC), and using reference values [reference doses (RfDs) or minimal risk levels (MRLs)] derived by the US Environmental Protection Agency (EPA), the NRC, and the US Agency for Toxic Substances and Disease Registry (ATSDR). Sewage sludge is routinely used in the agricultural setting as a means of replenishing the nutritional content of topsoil. However, as wastewater is processed in a treatment facility, compounds (e.g., industrial chemicals, fertilizers, heavy metals, etc.) may settle out and become concentrated in the sludge. Therefore, the concern of human exposure from sludge used in land application and cropland include: (1) uptake by plants and consumption by humans, (2) uptake by plants and consumption by livestock that may be consumed by humans, (3) direct ingestion of sludge-containing soil by grazing animals that may be consumed by humans, and (4) inadvertent ingestion of sludge-containing soil by humans. The paradigm outlined by the NRC for performing a risk assessment includes the following components: (1) hazard identification, (2) dose–response assessment, (3) exposure evaluation, and (4) risk characterization (NRC, 1983). The first two components (i.e., hazard identification and dose-response assessment) are known as a health assessment and entail an evaluation of the scientific literature for the types of effects that may be produced by a substance and the quantitative relationship between dose and effect(s). Collectively, the EPA and the ATSDR have completed health assessments and derived oral reference values on five PBDEs [i.e., tetra-, penta-, hexa-, octa-, and decabromodiphenyl ether (BDE)]. The NRC also performed a health assessment on decabromodiphenyl ether and developed an oral reference value (NRC, 2000). Each of these values may be used as a benchmark for use with exposure evaluation and risk characterization, and are provided in column five of Table 1. As mentioned previously, there are four primary exposure pathways that may be relevant for assessing human exposures to PBDEs from sludge-treated soils. Data are available on each. For