Exposure, Metabolism, and in Vitro Effects of Isopropylated and Tert-butylated Triarylphosphate Ester (ITP & TBPP) Flame Retardants and Plasticizers
Following the phase-out of polybrominated diphenyl ethers (PBDEs) in the early 2000s, organophosphate esters (OPEs) emerged as PBDE substitutes and have been applied to furniture foam, electronics, building materials, and some plastics to reduce their flammability. Although they have been used for quite some time in hydraulic fluids, isopropylated and tert-butylated triaryl phosphate esters (ITPs & TBPPs) have been more recently introduced as flame retardant (FR) replacements for the pentaBDE mixture in polyurethane foam (PUF). In addition to their use as FRs, ITPs and TBPPs are also used as plasticizers.
ITPs and TBPPs comprise a family of aryl organophosphate esters with varying degrees of isopropylation and tert-butylation. Individual ITP and TBPP isomers have been widely detected in indoor house dust, and recent biomonitoring studies demonstrate that human exposure to these compounds is widespread. Due to concerns about their persistence, bioaccumulation, and potential toxicity (P, B, & T), the U.S. Environmental Protection Agency (EPA) listed ITPs as one of five high priority chemicals fast-tracked for expedited risk assessment under the 2016 Toxic Substances Control Act (TSCA) reform.
As such, studying the exposure, metabolism, and in vitro effects of these compounds is especially timely. The hypothesis of this research dissertation is that ITP and TBPP isomers may exhibit some of the same P, B, & T properties that motivated the phase out of PBDEs. The main objectives of this research project were to generate meaningful data to fill gaps in our knowledge of ITP and TBPP isomers, and to contribute to the ongoing risk assessment of these compounds.
In the first aim of this thesis research, the maternal transfer of Firemaster® 550 (FM 550), a commercial mixture containing ITP isomers and brominated FRs, was investigated in dosed Wistar rats. Gestational and lactational transfer were examined separately, with dams orally exposed to 300 or 1000 µg of FM 550 for 10 consecutive days during gestation (gestational day [GD] 9-18) or lactation (postnatal day [PND] 3-12). Levels of parent compounds were measured in dam and pup urine. The two brominated components of FM 500, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis (2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP), underwent both gestational and lactational transfer. Triphenyl phosphate (TPHP) and ITPs were rapidly metabolized by the dams and were not detected in whole tissue homogenates. However, diphenyl phosphate (DPHP) and mono-isopropylphenyl phenyl phosphate (ip-PPP) were detected in urine from the dosed animals. This study was the first to confirm ip-PPP as a urinary metabolite of ITPs and establish a pharmacokinetic profile of FM 550 in a mammalian model.
In the second aim of this thesis research, the contribution of individual ITP and TBPP isomers was quantified in four commercial flame retardant mixtures: FM 550, Firemaster® 600 (FM 600), an ITP mixture, and a TBPP mixture. Findings suggested similarities between FM 550 and the ITP mixture, with 2-isopropylphenyl diphenyl phosphate (2IPPDPP), 2,4-diisopropylphenyl diphenyl phosphate (24DIPPDPP), and bis(2-isopropylphenyl) phenyl phosphate (B2IPPPP) being the most prevalent ITP isomer in both mixtures. FM 600 differed from FM 550 in that it contained TBPP isomers rather than ITP isomers. ITP and TBPP isomers were also detected and quantified in house dust standard reference material, SRM 2585, demonstrating their environmental relevance.
The third aim of this thesis research investigated phase I and II in vitro metabolism of TPHP, 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) at 1 and 10 µM doses using human liver subcellular fractions. Parent depletion and the formation of known metabolites, including DPHP, hydroxyl-triphenyl phosphate (OH-TPHP), ip-PPP, and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored via gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS). Tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP, accounting for up to 33% of the initial parent dose. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry (HRMS) and screened for in recently collected human urine. This study provided insight into recent human biomonitoring and epidemiological studies and contributed to our understanding of the biological fate of ITP and TBPP isomers.
Finally, the fourth aim of this thesis research evaluated ITPs, TBPPs, and related commercial mixtures for their effect on the activity of purified human liver carboxylesterase (hCE1). Four of the 15 OPEs tested had IC50 values lower than 100 nM, including TPHP, 2-ethylhexyl diphenyl phosphate (EHDPP), 4-isopropylphenyl diphenyl phosphate (4IPPDPP), and 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), as did four commercial flame retardant mixtures tested. Because hCE1 is critical for the activation of imidapril, an ACE-inhibitor prodrug prescribed to treat hypertension, the most potent inhibitors, TPHP and 4tBPDPP, and an environmentally relevant mixture (house dust) were further evaluated for their effect on imidapril bioactivation in vitro. TPHP and 4tBPDPP were potent inhibitors of hCE1-mediated imidapril activation (Ki = 49.0 and 17.9 nM, respectively), as were extracts of house dust (100 µg/ml), which caused significant reductions in imidapril activation. Combined, these data suggest that exposure to OPEs can affect pharmacotherapy.
Collectively and in context of other recently published findings, this thesis research suggests that ITPs and TBPPs may be regrettable substitutes for PBDEs.
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