Appendices of Screening Assessment Report

LD₅₀(oral, rat) greater than 50 000 mg/kg bw (International Bio-Research. Inc. 1967).
LD₅₀ (oral, mouse) = 3200 mg/kg (Gustafsson and Wallen 1988)

LC₅₀ (inhalation, rat) greater than 10,920 mg/m³ after 4 hr (Velsicol Chemical Corporation 1978e) (Decreased motor activity, eye squint, slight dyspnea and erythema).
LC₅₀ (inhalation, mouse) greater than 50 mg/L (50 000 mg//m³) after 8 hr withno signs of toxicity (Great Lakes Chemical Corp 1967a).

LD₅₀ (dermal, rabbit) greater than 10 000 mg/kg bw (Hill Top Research, Inc. 1966).

LD₅₀(oral, rat) greater than 5000 mg/kg bw (Abbott et al. 1981).

LD₅₀ (dermal, rabbit) greater than 2000 mg/kg (Abbott et al. 1981). Slight to moderate erythema and edema. The skin reaction decreased in severity and area over time. No gross pathological findings were observed.

LD₅₀(oral, rat) greater than 5000 mg/kg bw (Goldenthal and Dean 1974a)

LC₅₀ (inhalation, rat) greater than 12.5 mg/L (12500 mg/m³) (Goldenthal and Dean 1974a)

LD₅₀ (dermal, rabbit) greater than 2000 mg/kg (Goldenthal and Dean 1974a)

Lowest oral LOAEL = 700 mg/kg-bw per day based on slight enlargement of hepatocytes, inflammatory cell infiltration and focal necrosis of hepatocytes at this dose and higher (Tada et al. 2007). Male ICR mice were administered 0, 350, 700 or 1400 mg/kg-bw per day TBBPA via gavage for 14 consecutive days. Absolute and relative liver weight was significantly increased at the highest dose. 

Other oral NOAEL (rats, 28 days) = 250 mg/kg-bw per day. A gavage study with Wistar rats showed no significant hepatic effects in the liver (Szymanska et al. 2000). 

The EU RAR (2006) noted that most of the few repeated-dose studies by oral exposure were limited or poorly reported (IRDC 1972; Sato et al. 1996; Szymanska 1995; Frydrych and Szymanska 2001). More recent studies by Germer et al. (2006) reported no effects upon hepatic mRNA or microsomes in Wister rats when exposed to dietary concentrations equivalent to intakes of 0, 30, 100 or 300 mg/kg-bw per day for 28 days.

Lowest inhalation LOAEC (rats, 14 days) = 18 000 mg/m³ based on observations of local irritation in the upper respiratory tract (IRDC 1975). There were no toxicologically significant systemic effects (IRDC 1975).

Lowest dermal NOAEL (rabbits, 3 weeks) = 2500 mg/kg-bw per day. No adverse effects were observed (IRDC 1979).

Lowest oral NOAEL (rats, 90 days) = 100 mg/kg bw per day (The Dow Chemical Company 1975). No gross or histopathological lesions were observed in rats exposed by diet to 0, 0.3, 3, 30 or 100 mg/kg-bw per day for 90 days.

Other oral NOAEL (rats, 90 days) = 1000 mg/kg-bw per day (MPI Research 2002a). In a 13-week gavage study, rats were exposed to 0, 100, 300 or 1000 mg/kg-bw per day. There were no effects observed in either the functional observational battery or motor activity tests, no adverse histopathological changes in liver, thyroid, parathyroid or pituitary and no changes in serum levels of T3 or TSH.  Although there was a significant decrease in serum T4 in both sexes, in the absence of any other relevant thyroid-related effects, this was not considered adverse.

Negative: S.typhimurium strains (TA1535, TA 1537, TA1538, TA92 TA98, TA100) 0 to 10,000 μg/plate with and without metabolic activation (Litton Bionetics Inc 1976; Velsicol Chemical Corporation 1978a; Israel Institute for Biological Research 1978; Ethyl Corporation 1981; The Dow Chemical Company 1985; Mortelmans et al. 1986; Great Lakes Chemical Corporation 1986).

Negative: in Yeast strains Saccharomyces cerevisiae D3 and D4 at TBBPA concentrations of 0 to 500 μg/plate with or without metabolic activation (Velsicol Chemical Corporation 1978a; The Dow Chemical Company 1985).

Lowest oral LO(A)EL (mice) = 140.5-379.9 mg/kg-bw per day based upon enlargement of hepatocytes, very slight focal necrosis of hepatocytes, and decreased serum triglyceride (quantitative data not presented) levels in female offspring at this dose (Tada et al. 2006). There was also an increase in total serum cholesterol (quantitative data not presented) in male offspring at this dose. Pregnant ICR mice fed a diet of 0%, 0.01%, 0.1%, or 1.0% TBBPA (0, 15.7-42.1, 140.5-379.9, or 1639.7-4155.9 mg/kg bw) from GD 0 to weaning at PND 27. Serum concentrations of total-cholesterol and triglycerides were also affected in dams and offspring. No effects upon litter size, litter weight, total number of offspring male or female offspring weight.

The lowest benchmark dose, BMDL = 0.5 mg/kg-bw per day for increased F1 testis weight (critical effect dose of 1.4 mg/kg-bw per day) and increased F1 male pituitary weight (critical effect dose, 2.2 mg/kg-bw per day; BMDL, 0.6 mg/kg-bw per day). Wistar rats were administered TBBPA with calculated intakes of 0, 3, 10, 30, 100, 300, 1000 or 3000 mg/kg-bw per day in diet for 70 days (male) or 14 days (female) prior to mating and continuing during mating and throughout gestation and lactation (van der Ven et al. 2008; Lilienthal et al. 2008). There were no effects upon endpoints of reproduction. Other effects noted included delayed sexual development in females, and effects upon brainstem auditory evoked potentials. There were no exposure-related histopathological changes in the organs of the F1 animals. There were no effects upon sperm counts or morphology. There was no effect upon the immunisation response against sheep red blood cells in F1 males.  Another “major effect” was the developmentally induced increase of hearing latency at low frequency, with BMDLs of 7.8 and 8.4 mg/kg-bw per day for males and females, respectively. It is noted that concerns have been published concerning the methodology employed in this assay (Banasik et al. 2009; Strain et al. 2009; Lilienthal et al. 2009; van der Ven et al. 2009). A previous study was performed on male Wistar rats which were administered TBBPA with calculated intakes of 0, 30, 100, or 300 mg/kg-bw per day in diet for 28 days. The only effects were a decrease in circulating T4 and increased T3 levels in male rats.

Lowest oral LOAEL (rats) = 200 mg/kg bw per daybased on polycystic lesions associated with the dilation of the tubules in the kidneys of 2 of 6 males at this dose (Fukuda et al. 2004). Newborn rats were dosed by gavage from days 4 to 21 after birth, at doses of 0, 40, 200 or 600 mg/kg-bw per day. Effects on the kidney were observed at the two highest doses.  Diarrhea observed in some male and some females treated with 200 and 600 mg/kg bw. In the same study, five-week old rats were dosed at levels of 0, 2000 or 6000 mg/kg-bw per day for 18 days. No similar histopathological renal effects were observed. The EU RAR (2006) selected the NOAEL of 40 mg/kg bw per day for the purpose of risk characterization. However, it is considered that the effect on the kidneys is the result of “unconventional direct gavage administration of very high doses of TBBPA to such young animals and the immature metabolic capability and/or immature kidneys. Therefore, the relevance to human health of this isolated finding is considered questionable”.

Other oral LOEL = 100 mg/kg-bw per day based on a decrease in serum T4 levels in F0 and F1 offspring at this does and higher (MPI Research 2002b, 2003). Sprague Dawley rats were administered 0, 100, 200 or 1000 mg/kg-bw per day gavage for 10 weeks premating, 2 weeks mating and for females, throughout gestation and lactation. Serum T3 levels decreased significantly in F0 males in the 1000 mg/kg-bw dose group. No effects in either the F1 or F2 pups with regard to body weight, clinical findings, sex ratios, survival to weaning, macroscopic findings or organ weights. It was concluded that the effects in T3 and T4 levels were not toxicologically significant, as there was little impact on other parameters (MPI Research 2002b, 2003).

Other oral LOEL = 818.9-2129.2 mg/kg-bw per day based on decreased relative uterine weight in female offspring at post-natal week 11 at this dose (Saegusa et al. 2009). No other histopathological changes were observed in the uterus or any other organs examined. Pregnant Sprague-Dawley rats were exposed to dietary levels of 0, 100 ppm (9.5 – 22.9 mg/kg-bw per day), 1000 ppm (86.8 – 202.1 mg/kg-bw per day) or 10,000 ppm (818.9 – 2129.2 mg/kg-bw per day) from gestational day 10 to post natal day 20 after delivery (weaning). TBBPA did not alter normal brain development. Relative kidney weights decreased significantly at 1000 ppm dose level, but not at the higher dose in female offspring. There were no significant dose-related effects on T3, T4 or TSH. No effects upon number of implantation sites, number of live offspring or sex ratio (Saegusa et al. 2009).

A prepubertal exposure study examined the effects of TBBPA on susceptibility to thyroid tumours induced by a further exposure to DHPN or DMBA in Fisher 344 rats. Although the results of a complex exposure scenario are not taken into consideration in the assessment of TBBPA alone, the initial administration of 1% (1249 mg/kg-bw) TBBPA to dams from parturition to weaning (3 weeks) showed a statistically significant increase in thyroid weights and a decrease in relative liver weights (Imai et al. 2009). However, no histopathological changes were found in the liver and only one dam in 6 had diffuse follicular cell hyperplasia in the thyroid.

No developmental or neurotoxicological effects were observed at doses up to 10,000 mg/kg-bw per day and 1000 mg/kg bw per day, respectively in various studies (Velsicol Chemical Corporation 1978c; Noda et al. 1985; Eriksson et al. 1998, 2001; MPI Research 2001, 2002b, 2003; Hass et al. 2003).

Acute oral LOEL = 11.5 mg/kg bw based on a decrease in binding sites of the nicotinic ligand cytisine in frontal cortex, but not in the parietal cortex or hippocampus in male neonate mice.

Male NMRI mice (n=6-8) were given 11.5 mg TBBPA/kg bw (21 µmol) or 20% fat-emulsion vehicle/kg bw on PND 10 via a metal gastric-tube, as one single oral dose. The animals were killed 7 days after treatment and protein levels of CaMKII, GAP-43, synaptophysin and tau in hippocampus and cortex were measured using slot-blot analysis. [³H]-QNB binding (all subtypes of muscarinic receptors), [³H]-AFDX 384 binding (M2/M4 muscarinic receptors) and [³H]-cytisine binding (α4β2 nicotinic receptors) were assessed in the hippocampus, parietal and frontal cortex. The statistical evaluation was made using one-way ANOVA and pairwise testing using Newman–Keul’s post hoc test. TBBPA did not appear to affect the levels of proteins involved in maturation of the brain, neuronal growth or synaptogenesis in neonate mice. However, there was a decrease in binding sites of the nicotinic ligand cytisine in frontal cortex, (35.9 ± 10.4 pmol/g protein compared to 47.2 ± 7.7 pmol/g protein in controls), but not in the parietal cortex or hippocampus (Viberg and Eriksson 2011).

Oral LOEL = 86.8-202.1 mg/kg bw/day based on a transient increase in reelin-expressing interneurons in the dentate hilus at this dose and above in offspring at PND 20, but not at PND 77.

Pregnant Sprague–Dawley rats (n = 8/group) were exposed to 0, 100, 1000, or 10 000 ppm (0, 9.5 – 22.9, 86.8 – 202.1, 818.9 – 2129.2 mg/kg-bw per day) TBBPA in the diet from GD 10 through to day 20 after delivery (PND77). No major treatment-related changes were observed in dams during gestation and lactation. There were no dose-related changes in thyroid serum levels (See reproductive/developmental section above; Saegusa et al. 2009) and no effects on organ to body weight changes in the brain or the thyroid of offspring. A slight increase in apoptotic bodies in offspring (n ~ 20/sex/group) was observed at 818.9 – 2129.2 mg/kg-bw per day at PND 20, but this effect appeared reversible as there were very few apoptotic bodies at PND 77. An increase in reelin-expressing interneurons in the dentate hilus was observed at the mid and high doses of TBBPA, but again, these effects returned to control levels at PND77. There was an excess of mature neurons in the hilus later stages, but these effects were reversible. No changes were observed in the number of GAD67- immunoreactive cells in the highest dose groups compared with the untreated controls at both PND 20 and PND 77 nor any changes in EphA5- and Tacr3-immunoreactive cells in the hippocampal CA1 region (Saegusa et al. 2012).

Behavioural changes were observed in the two lowest dose groups of exposed male mice to 0, 0.1, 5 or 250 mg/kg-bw once by gavage, but were not considered to be treatment related. It is noted that the authors proposed that a compensation mechanism may account for the lack of a dose-response relationship (Nakajima et al. 2009).

Cytotoxicity: TBBPA appeared to be cytotoxic at low micromolar concentrations (LC₅₀ = 15 ± 4 µM) on SH-SY5Y human neuroblastoma cells, however the authors stated that it is unclear from this study if these results showed that TBBPA is neurotoxic (Al-Mousa and Michelangeli 2012). TBBPA caused activation of caspases (3/7) after the cells were exposed to TBBPA for 12 hours at a 1 to 5 µM concentration range. There was also a transient increase in intracellular [Ca²⁺] levels and reactive-oxygen-species (ROS) within these neuronal cells. Furthermore, TBBPA also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells (at 10 µM). Application of 3 and 10 µM of TBBPA for 12hrs caused increased b-amyloid peptide (Ab-42) processing and release from these cells with a few hours of exposure (Al-Mousa and Michelangeli 2012).

TBBPA was also found to be acutely cytotoxic in primary cultures of rat cerebellar granule cells after 30 minute exposures to 10^-50 µM of TBBPA (significant at 25 uM). According to the authors, TBBPA also induced an increase in intracellular Ca²⁺ concentrations, depolarization of mitochondria, and activation of ROS production (Zieminska et al. 2012).

Kitamura et al. (2005b) exposed ovariectomized B6C3F1 mice to 0, 20, 100, 300 and 500 mg/kg bw of TBBPA by ip injection for three days. Although the uterus to body weight ratio was significantly increased in all exposed groups, there was a poor dose-response.

Other oral LOEL: 150 mg/kg bw/day based on a decrease in T3-independent transcription activation of both Trh and Mc4r promoter genes in the hypothalamus of offspring on PND 2 (n greater than 10/group). Pregnant Swiss wild-type mice (n greater than 10/group) were administered 150 mg/kg bw of TBBPA daily via oral gavage from day 13 post conception for 7 days. The activity of Trh and Mc4r promoters was measured in the pup hypothalami using a reporter gene assay and TBBPA significantly reduced (P less than 0.001) T3-independent transcription from both Mc4r and Trh reporter constructs in pups. When pups were administered TBBPA in acute doses with one or two 2.1 g/kg injections of TBBPA, opposite effects were observed on T3-independent transcriptional activity of both promoters. In protocol no. 1, a single injection 48 h before sacrifice decreased Mc4r and Trh transcription in absence of T3 by 32.4% and 33.6% respectively. In contrast, administering TBBPA injections 48 and 24 h before sacrifice significantly increased transcription from both promoters: 28.7% and 37.5% for Mc4r and Trhconstructs, respectively (Decherf et al. 2010).

Other oral NOEL: 1000 mg/kg bw/day based on the lack of change in uterine weight in adult female mice. TBBPA was administered daily via oral gavage and subcutaneous injection for 7 days using C57BL/6J ovariectomized adult female mice (n = 6) in accordance with OECD Test Guideline No. 440. For detection of agonistic activity, control, 4 doses at a half-log ratio, and EE as a positive control were tested. For antagonistic activity, a control and the same 4 doses were administered with a reference dose of EE. The LOEL was defined as the lowest dose that induced significant change in uterine weight. Results from this study showed that TBBPA was negative for agonistic and antagonistic estrogenic responses by both routes of exposure using concentrations up to 1000 mg/kg bw/day (Ohta et al 2012).

Strong PR antagonist/Weak ERα agonist: Li et al. (2010) measured ER and PR-mediated transcription of β-galactosidase in vitro in reporter yeasts. TBBPA also exhibited the ability to reverse the estrogen-related receptor (ERR) inhibition induced by 4-hydrooxytamoxifen. Yeast strains were tested with increasing concentrations of TBBPA (1 x 10^-9to 1 x 10^-4 mol per L) for 2hrs.

Estrogenic activity of TBBPA was evident using ERE-luciferase reporter assay in MCF-7 breast cancer cells at 1x10^-6 to 1x10^-4 M. Anti-estrogenic acitivty was also measured in an E2 assay sysem in MCF-7 cells at 1x10^-5 M (Kitamura et al. 2005b).

Negative PR antagonist/ Negative ER agonist: No detectable antiprogestagenic or ER agonistic potency was measured in ER- and PR-CALUX assays in human breast cancer (ER) and human osteoblast (PR) cells at 10 and 12.5 µM concentrations, respectively (Hamers et al. 2006).  No ER agonistic/antagonistic effects were found by Riu et al. (2011) using HGELN, HGELN-ERα, HGELN-ERβ reporter cell lines (10^-9 to 10^-5M).

No effect was observed on cell growth in an E-screen assay in MCF-7 breast cancer cells even at a maximum concentration of 20 µM TBBPA did not induce estrogen receptor-mediated TFFl gene expression in vitro (Dorosh et al. 2010).

TBBPA did not show any estrogenic activity at the estrogen receptor alpha (ERα) ligand at concentrations between 10^-10 and 10^-5 M in an OECD guideline stably transfected transcriptional activation (STTA) assay (Lee et al. 2012).

Positive Inhibitor of E2 Sulfation: Hamers et al. (2006) reported that TBBPA was a potent inhibitor of E2 (estradiol) sulfation (IC = 0.016 uM) in an E2SULT assay.

Positive competitor of T4 binding to TTR: TBBPA was a potent T4 competitor in a TTR-binding assay (IC50 less than 0.1 µM) with a 1.6 times higher TTR-binding potency than the natural ligand T4 (Hamers et al. 2006).

Conflicting Thyroid Hormone agonist/antagonist:
An increase in growth hormone-releasing activity was observed from GH3 cells after addition of TBBPA at 1x10^-6 to 1x10^-4 M (Kitamura et al. 2005b). No antagonistic activity was found. Further work using GH3 cells (GH3.TRE-Luc) has shown that TBBPA acts as a very weak agonist (5% of T3-max) up to a concentration of 1 µM, but a slight antagonist at concentrations above 5 µM in the presence of 0.25 nM of T3 after only 24 hours of exposure (Freitas et al. 2010).

Administration of 25 µM TBBPA significantly suppressed TRβ activity (IC50 of 2.95 x 10^-5 M). Authors developed a TRβ-1 mediated reporter gene assay by transiently transfecting Gal4-fused thyroid hormone receptor (TR) expressing vector and the Gal4 response reporter structure pUAS-tk-luc into HepG2 cells (1, 10, 25, 50 and 100 µM). When treated alone, TBBPA could not induce the expression of luciferase which indicated that it could not activate TR (Sun et al. 2009).

Another TH-responsive luciferase–based reporter gene assay using the human hepatocarcinoma cell line HepG2 showed that TBBPA displayed agonistic effects at 10 µM, but was antagonistic when coincubated with T3 at 1 µM (concentrations ranged from 10^-4 to 10 µM for 24 h; Hofman et al. 2009).

TBBPA bound to the human TRα-LBD, activating transcription when applied alone (without T3) at 3 and 10µM. Further, TBBPA displaced physiological concentrations of T3 from TRα binding in HeLA cells using a reporter system based on fusion of the ligand-binding domain (LBD) from TRα to a GAL4 DNA– binding domain at 10 µM (Fini et al. 2012).

TBBPA prevented binding (or induced dissociation) of NcoRp, but failed to promote SRC2p binding, and even inhibited T3-induced SRC2p binding in a coactivator/corepressor peptide binding assay. Increasing concentrations (2–50 µM) of TBBPA were incubated with GST-LBD and FITC-labeled corepressor peptide (NcoRp) or coactivator peptide (SRC2p) in the presence and absence of T3 (cell-free) (Lévy-Bimbot et al. 2012).

At concentrations as high as 10 µM, TBBPA did not affect transcriptional activities of TRα and TRβ in any of the species studied (three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligatormississippiensis) and human (Homo sapiens).  In order to examine whether T3-stimulated activity was inhibited by TBBPA, T3 was added at a concentration that generated an EC₅₀ value for each receptor type (TRα: 4x10–10 M and TRb: 2x10–9 M). Results indicated that TBBPA inhibited T3-stimulated activation of TRα and TRβ from S. tropicalis andhuman cells at concentrations higher than 10µM (however, the cell viability after treatment with TBBPA was compromised at doses higher than 10 µM). Therefore, TBBPA can be evaluated at this concentration or less in this assay, which showed no effects on activity in human cells in vitro. Using this approach, authors noted that T3-induced transactivity of the O. latipes TRα was inhibited by 10 µM TBBPA, but this compound at this concentration did not antagonize TRβ activity   (Oka et al. 2012).

Deiodinase Inhibition: Almost complete inhibition of DI activity was observed at the highest dose tested (2.1µM) for TBBPA. A dose-response relationship was shown for TBBPA in inhibition of the formation of rT3 from T4. Inhibition of 3,3’-T2 formation was also observed at 1.9 µM. Thyroxine (T4) and reverse triiodothyronine (rT3) deiodination kinetics were measured by incubating pooled human liver microsomes with T4 or rT3 and monitoring the production of T3, rT3, 3,3’-diiodothyronine, and 3-monoiodothyronine by liquid chromatography tandem mass spectrometry (Butt et al. 2011).

Equivocal AR antagonist: Weak antiandrogenic activity in MDA-kb2 cells in vitro between 10 and 50 µM for 24 hrs followed by a luciferase assay (Christen et al. 2010). Li et al. (2010) and Hamers et al. (2006) did not find any effect on AR activity in yeast cells (concentration not listed) or in human osteoblast cells at 10 µM concentrations using an AR-CALUX assay, respectively, after administration of TBBPA. No antiandrogenic activity was found in an AR responsive luciferase reporter gene system in NIH3T3 cells at 1x10^-11 to 1x10^-9 M concentration range (Kitamura et al. 2005b).

Negative aromatase activity: TBBPA did not inhibit or induce aromatase (CYP19) activity and was not cytotoxic at concentrations of 2.5 µM and 7.5 µM in H295R human adrenocortical carcinoma cells after 24 hr incubation (Cantón et al. 2005).

Immunotoxicity in vitro: Exposure of 0 to 10 µM of TBBPA to human natural killer cells decreases the lytic function at concentrations as low as 0.5 µM after 24 hrs exposure and 1.0 µM after 1 hr exposure to TBBPA (Kibakaya et al. 2009).  A follow up study showed that the expression of cell-surface proteins CD2, CD11a, CD16, CD18 needed for attachment of NK cells to target cells was decreased after exposure to 5 µM TBBPA for 24 hr (Hurd and Whalen 2011). NK cells were exposed to TBBPA (0, 1, 2.5, 5 and 10 µM) for 24h, 48h, and 6 days or for 1 hr followed by 24h, 48 hr, and 6 days without TBBPA. Each experiment was completed four times using cells prepared from different blood donors. CD16 expression was decreased by greater than 35%, CD11a by 16%, and CD18 and CD56 by ~ 20%. Expression of CD18 was also decreased (14%) at 2.5 µM. Cell viability was compromised at 10 µM TBBPA (at all timepoints) and at 5 µM after 48 hrs and not evaluated. Expression of CD16 and CD56 proteins were similar at 24 and 48 hrs at 2.5 µM (Hurd and Whalen 2011).

Han et al. (2009) reported that exposure to TBBPA (0- 50 µM) induced COX-2 transcription and proinflammatory cytokine expression through the P13-K/Akt/MAPK signalling pathway mediated via increased NF-κB and AP-1 activation in murine macrophages in vitro.

Immunotoxicity in vitro: Mice splenocytes incubated with a TBBPA allyl ether (10 µmol/L) exhibited significantly reduced expression of the interleukin-2 receptor α chain (CD25), an antigen necessary for the production of activated T cells during an immune response (Pullen et al. 2003).

Exposure of 2 uM and higher concentrations of TBBPA to human neutrophil granulocytes enhanced reactive oxygen species (ROS) production in a concentration dependent manner (Reistad et al. 2005).

Mice splenocytes incubated with a TBBPA (3 µmol/L) exhibited significantly reduced expression of the interleukin-2 receptor α chain (CD25), an antigen necessary for the production of activated T cells during an immune response (Pullen et al. 2003).

TBBPA showed no cytotoxic effect on either splenocytes or bone marrow-derived dendritic cells (BMDCs) from atopic prone NC/Nga mice. After exposure to TBBPA, cell surface molecule expression and cytokine production was measured using WST-1 assay, FACS and ELISA (three individual cultures obtained from three animals and two or three independent experiments were repeated). Spenocytes were exposed to 0.01–10 µg/ml TBBPA for 24 hrs and BM cells were exposed to 0.001 – 1 µM TBBPA for 6 days and BMDCs were collected. TBBPA did increase MHC class II and CD86 expression, and T-cell receptor (TCR) expression in splenocytes (1 and 10 µg/ml) and increased interleukin (IL)-4 production (but a chemical-dependent impact pattern was observed). There was no effect on expression of MHC class II, CD80, CD86, CD11c and DEC205) and chemokine production after 24-h exposureafter TBBPA exposure in BMDCs (Koike et al. 2012). 

Immunotoxicity in vivo: Exposure in mice to 1% TBBPA in the diet for 28 days (1887 mg/kg-bw per day) demonstrated that host immunity to respiratory syncytial virus was affected in lung and BALF samples while systemic immunity was not affected when spleen samples were examined (Watanabe et al. 2010).

Hepatoxicity in vitro: TBBPA competitively inhibited the binding of [³H]-rosiglitazone to PPARγ with a half maximal inhibitory concentration (IC50) of 0.7 µM. HeLa cells transiently transfected with (GALRE)5-βglobin-luciferase and pSG5-GAL4-PPARγ (human and zebrafish) or (PPRE)3-TK-luciferase and pSG5-PPARγ (Xenopus laevis) plasmids were incubated with 10 μM TBBPA or 1 μM rosiglitazone (Rosi) to assess their agonist potential on PPARγ. The binding affinities to PPARγ by competitive binding assays with [³H]-rosiglitazone were measured  (0.001–30 μM) and the impact of TBBPA on adipocyte differentiation using NIH3T3-L1 cells was investigated.  At 10 μM, TBBPA also induced adipogenesis, whereas co-treatment with CD5477 inhibited the adipogenic action of TBBPA, indicating that TBBPA mediates adipogenesis via PPARγ (Riu et al 2011).

Alternatively, when the ability of TBBPA was tested to activate human PXR or mouse PXR using a transfection assay, results were negative (Siu et al. 2012). HepG2 cells were transfected with either full-length hPXR together with CYP3A4-luc reporter or full-length mPXR together with (CYP3A2)3-luc reporter and CMX-ß-galactosidase control plasmid. Cells were treated with DMSO (control) or TBBPA for 24 h at 0-20 µM concentrattions.