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Endpoint:
biochemical or cellular interactions
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Special investigation
Principles of method if other than guideline:
Several brominated flame retardants were administrated to chicken embryonic hepatocytes (CEH) to determine cytotoxicity and changes in relative mRNA abundance of 11 genes involved in xenobiotic metabolism, lipid metabolism and thyroid hormone (TH) homeostasis by real-time reverse transcription-PCR.

GLP compliance:
no
Type of method:
in vitro
Specific details on test material used for the study:
purity > 98 %
Species:
other: not applicable - in-vitro assay
Strain:
other: not applicable - in-vitro assay
Sex:
not specified
Route of administration:
other: not applicable - in-vitro assay
Vehicle:
other: not applicable - in-vitro assay
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
not applicable - in-vitro assay
Frequency of treatment:
not applicable - in-vitro assay
Post exposure period:
not applicable - in-vitro assay
Remarks:
Doses / Concentrations:
0.001-30 µM
Basis:
nominal conc.
No. of animals per sex per dose:
not applicable - in-vitro assay
Control animals:
other: not applicable - in-vitro assay
Details on results:
Several brominated flame retardants were administrated to chicken embryonic hepatocytes (CEH) to determine cytotoxicity and changes in relative mRNA abundance of 11 genes involved in xenobiotic metabolism, lipid metabolism and thyroid hormone (TH) homeostasis by real-time reverse transcription-PCR.
-> None of the gene targets were responsive to TBPH exposure in CEH.
Executive summary:

Several brominated flame retardants were administrated to chicken embryonic hepatocytes (CEH) to determine cytotoxicity and changes in relative mRNA abundance of 11 genes involved in xenobiotic metabolism, lipid metabolism and thyroid hormone (TH) homeostasis by real-time reverse transcription-PCR.

 None of the gene targets were responsive to TBPH exposure in CEH.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: special investigation
Principles of method if other than guideline:
The potential of 11 brominated flame retardants to interfere with estrogenic and androgenic pathways was examined using two yeast reporter-gene assays. The following activities were investigated using recombinant Saccharomyces cerevisiae strains expressing beta-galactosidase or luciferase in response to estrogen or androgen exposure: estrogen-like activity; anti-estrogen like activity; androgen like activity and anti-androgen like activity.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: endocrine activity
Specific details on test material used for the study:
TBPH, 99.5%, GC/MS
Species:
other: not applicable - in-vitro assay
Strain:
other: not applicable - in-vitro assay
Route of administration:
other: not applicable - in-vitro assay
Vehicle:
other: not applicable - in-vitro assay
Duration of treatment / exposure:
not applicable - in-vitro assay
Frequency of treatment:
not applicable - in-vitro assay
Post exposure period:
not applicable - in-vitro assay
No. of animals per sex per dose:
not applicable - in-vitro assay
Control animals:
other: not applicable - in-vitro assay
Details on results:
TBPH tested at 2 µg/ml did not show growth inhibition. No hormonal like activity was observed in all tests reported (i.e. no estrogenic activity; no androgenic activity, no anti-estrogenic activity and no anti-androgenic activity).

TBPH tested at 2 µg/ml did not show growth inhibition. No hormonal like activity was observed in all tests reported (i.e. no estrogenic activity; no androgenic activity, no anti-estrogenic activity and no anti-androgenic activity).

Executive summary:

The potential of 11 brominated flame retardants to interfere with estrogenic and androgenic pathways was examined using two yeast reporter-gene assays. The following activities were investigated using recombinant Saccharomyces cerevisiae strains expressing beta-galactosidase or luciferase in response to estrogen or androgen exposure: estrogen-like activity; anti-estrogen like activity; androgen like activity and anti-androgen like activity.

 TBPH tested at 2 µg/ml did not show growth inhibition. No hormonal like activity was observed in all tests reported (i.e. no estrogenic activity; no androgenic activity, no anti-estrogenic activity and no anti-androgenic activity).

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: special investigation
Principles of method if other than guideline:
The effect of TBPH and other brominated flame retardants on steroidogenesis in primary porcine testicular cells was investigated. The primary cell culture system derived from pig testes contained androgen synthesizing Leydig cells, supportive Sertoli cells and andrenal-like cells derived from the intestitium of testis. Medium was collected after exposure of the cells to the test chemicals (i.e. 0.15 and 15 mg/l TBPH) for 48 hours. Hormones (testosterone (T) and estradiol (E2)) were extracted and quantification was done by ELISA. Additionally, the expression of key enzymes of steroidogenesis was examined.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: endocrine activity
Species:
other: neonatal piglets
Strain:
not specified
Sex:
male
Route of administration:
other: not applicable - in-vitro assay
Vehicle:
other: not applicable - in-vitro assay
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
not applicable - in-vitro assay
Frequency of treatment:
not applicable - in-vitro assay
Post exposure period:
not applicable - in-vitro assay
Remarks:
Doses / Concentrations:

Basis:
other: not applicable - in-vitro assay
No. of animals per sex per dose:
not applicable - in-vitro assay
Control animals:
other: not applicable - in-vitro assay
Details on results:
Increased E2 and T production was observed for both concentrations. In the case of T, no dose-response relationship was observed. For E2, a clear trend was also missing, because 0.15 mg TBPH/L resulted in 4.6-fold higher E2 production whereas at the 100 fold higher concentration of 15 mg TBPH/L E2 production was only slightly augmented to 5.3-fold over control.

Exposure of primary cells to TBPH did not affect expression of StAR, CYP17A1, CYP21A2, 3 βHSD, or 17β-HSD m-RNA at either of the concentrations tested. Expression of CYP11A1 was 3.5-fold and 6.6-fold greater in cells exposed to 0.15 and 15.0 mg TBPH/L, respectively, relative to that of solvent controls. No change in expression of CYP19A1 was observed in cells exposed to 0.15 mg TBPH/L, though expression of mRNA for CYP19A1 was 3.3-fold greater when the cells were exposed to 15 mg TBPH/L.

Overall, the validity and relevance of this in-vitro test for human risk assessment is restricted since only 2 different concentrations (0.15 and 15.0 mg TBPH/L) were measured and no detectable TBPH was reported in serum in an in vivo rat toxicokinetic study after single dosing with mg TBPH/kg (Silva MJ, Hilton D, Furr J, Gray LE, Preau JL, Calafat AM, Ye X, Quantification of tetrabromo benzoic acid and terabromo phthalic acid in rats exposed to the flame retardant Uniplex FRP-45, Arch Toxicol DOI 10.1007/s00204-015-1489-6, published online: 25 March 2015).

Executive summary:

The effect of TBPH and other brominated flame retardants on steroidogenesis in primary porcine testicular cells was investigated. The primary cell culture system derived from pig testes contained androgen synthesizing Leydig cells, supportive Sertoli cells and andrenal-like cells derived from the intestitium of testis. Medium was collected after exposure of the cells to the test chemicals (i.e. 0.15 and 15 mg TBPH/L) for 48 hours. Hormones (E2 and T) were extracted and quantification was done by ELISA. Additionally, the expression of key enzymes of steroidogenesis was examined.

Increased E2 and T production was observed for both concentrations. In the case of T, no dose-response relationship was observed. For E2, a clear trend was also missing, because 0.15 mg TBPH/L resulted in 4.6-fold higher E2 production whereas at the 100 fold higher concentration of 15 mg TBPH/L E2 production was only slightly augmented to 5.3-fold over control.

Exposure of primary cells to TBPH did not affect expression of StAR, CYP17A1, CYP21A2, 3 βHSD, or 17β-HSD m-RNA at either of the concentrations tested. Expression of CYP11A1 was 3.5-fold and 6.6-fold greater in cells exposed to 0.15 and 15.0 mg TBPH/L, respectively, relative to that of solvent controls. No change in expression of CYP19A1 was observed in cells exposed to 0.15 mg TBPH/L, though expression of mRNA for CYP19A1 was 3.3-fold greater when the cells were exposed to 15 mg TBPH/L.

Overall, the validity and relevance of this in-vitro test for human risk assessment is restricted since only 2 different concentrations (0.15 and 15.0 mg TBPH/L) were measured and no detectable TBPH was reported in serum in an in vivo rat toxicokinetic study after single dosing with mg TBPH/kg (14).

Endpoint:
endocrine system modulation
Type of information:
other: read-across from Firemaster 550® (FM 550) contains 4 different components (see details on test material)
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Special investigation
Principles of method if other than guideline:
The accumulation and endocrine disrupting effects of the flame retardant mixture Firemaster® 550 in rats was investigated in an exploratory study.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
other: endocrine activity
Species:
rat
Strain:
Wistar
Sex:
female
Route of administration:
oral: feed
Vehicle:
other: AIN-76A Rodent Diet Test Tabs, Test Diet, Richmond, IN
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Daily oral exposure spanned GD 8 through weaning. On PND 21, pups were weaned.
Frequency of treatment:
Daily
Post exposure period:
On PND 21, s subset of animals and all dams were sacrificed.
Offspring were weighed on PNDs 1, 10, 21, 120, 180, and the day of sacrifice (PND 220).
Remarks:
Doses / Concentrations:
0, 100 or 1000 µg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
Three dams per group
Control animals:
yes, concurrent vehicle
Details on results:
Gestation length, liter size, and litter composition did not significantly differ between groups. In the male offspring, significant group differences in body weight became apparent by PND 10 and persistent through weaning on PND 21 with the males in the high-dose group weighing significantly more than controls. Among the female offspring, group differences in body weight were significantly at PND 21 with high-dose group weighing significantly more than controls. No significant differences in dam weight at sacrifice were observed.

Adipose, liver, and muscle tissue samples were collected from each dam on PND 21 and analyzed for TBPH. TBPH was only detected in dam liver tissues. TBMEHP was not detected in any tissues.

Total serum T4 levels were significantly higher in the high-dose exposed dams compared to control dams. T4 levels were not significantly higher compared to controls. There was no statistically significant main effect of the experimental group on total T3 levels in dam serum. There were also no statistically significant differences in T4 or T3 levels in pup serum on PND 21. CYP1A1 activity was also not significantly different among groups on PND 21.

Baseline blood glucose levels were significantly altered by exposure in males, but not females, with elevated levels in the high-dose group compared to same sex controls. Significant main effects on blood glucose levels during the glucose challenge test were only observed in females.
At 30 min, the high-dose females had significantly higher blood glucose levels compared to controls; an effect which was no longer significant at 60 min. AUC was higher in the high-dose females compared to controls, but the effect did not reach statistical significance.

No significant differences were observed in female heart weight and LV thickness, nor in male heart weight. In males, LV wall thickness was significantly increased, with a significantly increased LV wall thickness in males exposed to the high dose.

Altogether, due to the low number of animals (3 per dose group, one of the dams in the high-dose group failed to litter), the statistical relevance of the results is insufficient. Additional, some of the effects were seen only temporarily. Therefore, the results of this exploratory study are not suitable to demonstrate clear endocrine disrupting effects.

However, it should be mentioned, that in this study in Wistar rats TBMEHP, the primary metabolite of TBPH, was not detected in any tissue examined.

In the publication it was reported, that EPA entered into a modified consent order with Chemtura and required further testing of FM 550. Specifically, they required a two generation reproductive study (MPI Research Study 1038– 008; “CN-2065: An Oral Two-Generation Reproduction and Fertility Study in Rats”) and a developmental toxicity study (MPI Research Study 1038–006; CN-2065: Prenatal Developmental Toxicity Study in Rats”). Exposures in both toxicity studies were conducted at doses ranging from 15 to 300 mg/kg/day using oral gavage. In both studies, the no observable adverse effects level (NOAEL) was set at 50 mg/kg/day according to Patisaul et al., 2013 (14).

The findings of the exploratory study of Patisaul et al. were not confirmed by the Oral Two-Generation Reproduction and Fertility Study in Rats and the Prenatal Developmental Toxicity Study in Rats with FM 550.

Executive summary:

TBPH ( bis(2-ethylhexyl) tetrabromophthalate) is a component of Firemaster 550® (FM 550). FM 550 contains 4 different components: triphenyl phosphate (TPP), a mixture of isopropylated triphenylphosphate isomers (ITPs), TBB and TBPH. According to Patisaul et al. (14) the total sum of TBB and TBPH in FM 550 is assumed to be 50% (no further data).

Three dams (Wistar rats) per group received doses of 0, 100 or 1000 µg of the test substance per day in diet (Patisaul et al. (16)). Daily oral exposure spanned GD 8 through weaning. Day of birth was designated postnatal day (PND) 0. Sex ratio and body weights of each litter were obtained on PND 1, 10, and 21.On PND 21, pups were weaned. On PND 21, s subset of animals and all dams were sacrificed. Animals weight was recorded, and blood, muscle, liver, and gonadal adipose tissue was collected. The remaining offspring were sacrificed approx. PND 220. On PND 21a subset of animals and all dams were sacrificed. Animal weight was recorded, and blood, muscle, liver and gonadal adipose tissue were collected. The remaining offspring were sacrificed approx. PND 220 and the same tissues collected as for the juveniles plus heart. Total (free and protein bound) thyroxine (T4) and total triiodothyronine (T3) were measured in serum from individual dams and pups (pooled) on PND 21 and in individual pups at 7 months of age. CYP1A1 activity was measured in liver microsomal fractions. Offspring were weighed on PNDs 1, 10, 21, 120, 180, and the day of sacrifice. The glucose challenge test was performed at 17 weeks of age (approx. PND 120). Hearts from adult offspring were weighed and left ventricular (LV) free wall thickness measured. Results: Gestation length, liter size, and litter composition did not significantly differ between groups. In the male offspring, significant group differences in body weight became apparent by PND 10 and persistent through weaning on PND 21 with the males in the high-dose group weighing significantly more than controls. Among the female offspring, group differences in body weight were significantly at PND 21 with high-dose group weighing significantly more than controls. No significant differences in dam weight at sacrifice were observed. Adipose, liver, and muscle tissue samples were collected from each dam on PND 21 and analyzed for TBPH. TBPH was only detected in dam liver tissues. TBMEHP was not detected in any tissues. Total serum T4 levels were significantly higher in the high-dose exposed dams compared to control dams. T4 levels were not significantly higher compared to controls. There was no statistically significant main effect of the experimental group on total T3 levels in dam serum. There were also no statistically significant differences in T4 or T3 levels in pup serum on PND 21. CYP1A1 activity was also not significantly different among groups on PND 21. Baseline blood glucose levels were significantly altered by exposure in males, but not females, with elevated levels in the high-dose group compared to same sex controls. Significant main effects on blood glucose levels during the glucose challenge test were only observed in females. At 30 min, the high-dose females had significantly higher blood glucose levels compared to controls; an effect which was no longer significant at 60 min. AUC was higher in the high-dose females compared to controls, but the effect did not reach statistical significance. No significant differences were observed in female heart weight and LV thickness, nor in male heart weight. In males, LV wall thickness was significantly increased, with a significantly increased LV wall thickness in males exposed to the high dose. Altogether, due to the low number of animals (3 per dose group, one of the dams in the high-dose group failed to litter), the statistical relevance of the results is insufficient. Additional, some of the effects were seen only temporarily. Therefore, the results of this exploratory study are not suitable to demonstrate clear endocrine disrupting effects. However, it should be mentioned, that in this study in Wistar rats TBMEHP, the primary metabolite of TBPH, was not detected in any tissue examined. In the publication it was reported, that EPA entered into a modified consent order with Chemtura and required further testing of FM 550. Specifically, they required a two generation reproductive study (MPI Research Study 1038– 008; “CN-2065: An Oral Two-Generation Reproduction and Fertility Study in Rats”) and a developmental toxicity study (MPI Research Study 1038–006; CN-2065: Prenatal Developmental Toxicity Study in Rats”). Exposures in both toxicity studies were conducted at doses ranging from 15 to 300 mg/kg/day using oral gavage. In both studies, the no observable adverse effects level (NOAEL) was set at 50 mg/kg/day according to Patisaul et al., 2013 (14). The findings of the exploratory study of Patisaul et al. were not confirmed by the Oral Two-Generation Reproduction and Fertility Study in Rats and the Prenatal Developmental Toxicity Study in Rats with FM 550.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Special investigation
Principles of method if other than guideline:
The in-vitro metabolism of the brominated flame retardant TBPH (bis(2-ethylhexyl) tetrabromophthalate) was examined.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
basic toxicokinetics
Specific details on test material used for the study:
TBPH (99% purity) were purchased as neat solutions from AccuStandard, Inc. (New Haven, CT, USA).
Species:
other: not applicable - in-vitro assay
Strain:
other: not applicable - in-vitro assay
Sex:
not specified
Route of administration:
other: not applicable - in-vitro assay
Vehicle:
other: not applicable - in-vitro assay
Duration of treatment / exposure:
not applicable - in-vitro assay
Frequency of treatment:
not applicable - in-vitro assay
Post exposure period:
not applicable - in-vitro assay
No. of animals per sex per dose:
not applicable - in-vitro assay
Details on results:
In experiments with human liver microsomes (HLM), a significant loss of TBPH was not observed, and no metabolites were detected by GC/MS analysis of the sample extracts. An LC/MS-MS method was developed to monitor mono(2-ethylhexyl) tetrabromophthalate (TBMEHP), a potential hydrolysis metabolite of TBPH (Figure 1). After a 6-h incubation with HLM, TBMEHP was not detected as a metabolite of TBPH, and no significant loss of TBPH was observed. However, TBPH was slowly metabolized to form TBMEHP in the presence of 0.1mg/mL of porcine hepatic carboxylesterase (PCE). This reaction was monitored at multiple time points up to 6 h and maintained linearity at an approximate rate of 1.08 pmol/min/mg esterase.

In a previous study (Niino, T., Ishibashi, T., Ishiwata, H., Takeda, K., and Onodera, S. (2003) Characterization of human salivary esterase in enzymatic hydrolysis of phthalate esters. J. Health Sci. 49, 76−81). With PCE, DEHP (50 μM) was metabolized to form MEHP at a rate of 127 pmol/min/mg protein. This rate was approximately 100 times faster than the hydrolysis of TBPH observed in this study (1.08 pmol/min/mg protein).
The prominent difference between the metabolic hydrolysis of DEHP and TBPH may be a result of steric hindrance by the fully brominated phenyl ring of TBPH.

Overall, no metabolites of TBPH were observed with HLM. From this study there is no indication that TBPH is degradated to TBMEHP in vivo. In an in-vitro experiment the hydrolysis rate of TBPH with PCE is by factor 100 slower compared with DEHP.

Executive summary:

The in-vitro metabolism of the brominated flame retardant TBPH was examined.

In experiments with human liver microsomes (HLM), a significant loss of TBPH was not observed, and no metabolites were detected by GC/MS analysis of the sample extracts. An LC/MS-MS method was developed to monitor mono(2-ethylhexyl) tetrabromophthalate (TBMEHP), a potential hydrolysis metabolite of TBPH. After a 6-h incubation with HLM, TBMEHP was not detected as a metabolite of TBPH, and no significant loss of TBPH was observed. However, TBPH was slowly metabolized to form TBMEHP in the presence of 0.1mg/mL of porcine hepatic carboxylesterase (PCE). This reaction was monitored at multiple time points up to 6 h and maintained linearity at an approximate rate of 1.08 pmol/min/mg esterase.

In a previous study (cited in (13)) with PCE, DEHP (50 μM) was metabolized to form MEHP at a rate of 127 pmol/min/mg protein. This rate was approximately 100 times faster than the hydrolysis of TBPH observed in this study (1.08 pmol/min/mg protein).

The prominent difference between the metabolic hydrolysis of DEHP and TBPH may be a result of steric hindrance by the fully brominated phenyl ring of TBPH.

Overall, no metabolites of TBPH were observed with HLM. From this study there is no indication that TBPH is degradated to TBMEHP in vivo. In an in-vitro experiment the hydrolysis rate of TBPH with PCE is by factor 100 slower compared with DEHP.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Special investigation
Principles of method if other than guideline:
The endocrine activity of TBPH (bis(2-ethylhexyl) tetrabromophthalate) and other flame retardants was examined using the YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: endocrine activity
Specific details on test material used for the study:
Bis-(2-ethylhexyl) tetrabromophthalate (TBPH) was obtained from Waterstone Technology (Indiana, United States. Purity is reported to be >95% pure by the manufacturer.
Species:
other: not applicable - in-vitro assay
Strain:
other: not applicable - in-vitro assay
Sex:
not specified
Route of administration:
other: not applicable - in-vitro assay
Vehicle:
other: not applicable - in-vitro assay
Duration of treatment / exposure:
not applicable - in-vitro assay
Frequency of treatment:
not applicable - in-vitro assay
Post exposure period:
not applicable - in-vitro assay
No. of animals per sex per dose:
not applicable - in-vitro assay
Details on results:
YES/YAS bioassays:
Estrogenic and androgenic activities of TBPH were measured in recombinant Saccharomyces cerevisiae strains expressing beta-galactosidase in response to estrogen or androgen exposure:
Anti-estrogenic and anti-androgenic activities of TBPH was measured by reduction in activity of beta-galactosidase in yeast cells in the presence of 17 beta-estradiol (E2), and dihydrotestosterone (DHT), respectively.
-> TBPH caused no estrogen-like or androgen-like potencies in the YES/YAS bioassays.
-> TBPH produced anti-androgenic effects at 0.03 – 1500 mg/L. Beta-galactosidase expression was slightly but significantly reduced from 0.03 – 300 mg/L. At the highest concentration, 1500 mg/L, a response comparable to the positive control DHT was observed.
-> TBPH produced slight anti-estrogenic effects at 0.003 – 0.3 mg/L; but no effects were observed at higher concentrations of 3 – 150 µg/L.

H295R steroidogenesis assay:
H295Rcells were dosed with TBPH ranging from 1.5 to 30 mg/L. Forskolin, a strong inducer of both E2 and T production, and prochloraz, a strong inhibitor of both E2 and testosterone (T) production, were used as controls in the H295R steroidogenesis assay.
In the H295R steroidogenesis assay TBPH showed an increase of E2 production and a weak increase in T production. However, no dose dependent trend was observed

H4IIE-luc transactivation reporter gene assay:
The H4IIE-luc cellular assay is derived from rat hepatoma cells which have been stably transfected with a luciferase gene under control of a dioxin-responsive element. Cells were incubated for 24 h prior to dosing. Test and control wells were dosed with 1% per well volume of TBPH prepared in DMSO and luciferase activity was measured. The following concentrations of the test compound were used: 0.75, 1.5, 3, 15, 30, 150 mg TBPH/L. A 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) standard curve was included in each plate to control for inter-plate variability.
-> TBPH caused no TCDD-like potencies in the H4IIE-luc bioassay.

The relevance of the reported in vitro observations for human risk assessment is limited because the study was not conducted according to scientifically validated methods and the documentation is limited with respect to study design and results. In addition the concentrations used in these artificial in vitro tests are well above serum concentration reported in the toxicokinetic in vivo study in rats by Silva et al. 2015. In this study Sprague-Dawley rats were dosed with 500 mg TBPH/kg and serum concentrations were measured after 48 hours. No TBPH or oxidative metabolites similar to those formed by the DEHP were reported by the authors in urine or serum; no detection limit mentioned. Mean serum levels of the main metabolite 2,3,4,5-tetrabromo phthalic acid (TBPA) was 0.05 ± 0.03 mg/L (14); a concentration well below the concentrations used in the in vitro studies by Sauders et al. 2013.

Overall, based on the publication by Saunders et al. 2013 it cannot be concluded that TBPH causes any human risk assessment relevant endocrine activity.

Executive summary:

The endocrine activity of TBPH and other flame retardants was examined using the YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay.

YES/YAS bioassays:

Estrogenic and androgenic activities of TBPH were measured in recombinant Saccharomyces cerevisiae strains expressing beta-galactosidase in response to estrogen or androgen exposure:

Anti-estrogenic and anti-androgenic activities of TBPH was measured by reduction in activity of beta-galactosidase in yeast cells in the presence of 17 beta-estradiol (E2), and dihydrotestosterone (DHT), respectively.

 TBPH caused no estrogen-like or androgen-like potencies in the YES/YAS bioassays.

 TBPH produced anti-androgenic effects at 0.03 – 1500 mg/L. Beta-galactosidase expression was slightly but significantly reduced from 0.03 – 300 mg/L. At the highest concentration, 1500 mg/L, a response comparable to the positive control DHT was observed.

 TBPH produced slight anti-estrogenic effects at 0.003 – 0.3 mg/L; but no effects were observed at higher concentrations of 3 – 150 µg/L.

H295R steroidogenesis assay:

H295Rcells were dosed with TBPH ranging from 1.5 to 30 mg/L. Forskolin, a strong inducer of both E2 and T production, and prochloraz, a strong inhibitor of both E2 and testosterone (T) production, were used as controls in the H295R steroidogenesis assay.

In the H295R steroidogenesis assay TBPH showed an increase of E2 production and a weak increase in T production. However, no dose dependent trend was observed

H4IIE-luc transactivation reporter gene assay:

The H4IIE-luc cellular assay is derived from rat hepatoma cells which have been stably transfected with a luciferase gene under control of a dioxin-responsive element. Cells were incubated for 24 h prior to dosing. Test and control wells were dosed with 1% per well volume of TBPH prepared in DMSO and luciferase activity was measured. The following concentrations of the test compound were used: 0.75, 1.5, 3, 15, 30, 150 mg TBPH/L. A 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) standard curve was included in each plate to control for inter-plate variability.

 TBPH caused no TCDD-like potencies in the H4IIE-luc bioassay.

The relevance of the reported in vitro observations by Saunders et al. 2014 for human risk assessment is limited because the study was not conducted according to scientifically validated methods and the documentation is limited with respect to study design and results. In addition the concentrations used in these artificial in vitro tests are well above serum concentration reported in the toxicokinetic in vivo study in rats by Silva et al. 2015. In this study Sprague-Dawley rats were dosed with 500 mg TBPH/kg and serum concentrations were measured after 48 hours. No TBPH or oxidative metabolites similar to those formed by the DEHP were reported by the authors in urine or serum; no detection limit mentioned. Mean serum levels of the main metabolite 2,3,4,5-tetrabromo phthalic acid (TBPA) was 0.05 ± 0.03 mg/L (14); a concentration well below the concentrations used in the in vitro studies by Sauders et al. 2013.

Overall, based on the publication by Saunders et al. 2013 it cannot be concluded that TBPH causes any human risk assessment relevant endocrine activity.

Endpoint:
specific investigations: other studies
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: QSAR Toolbox Report 3.3.5 with the profile of bis(2-ethylhexyl) tetrabromophthalate (CAS No 26040-51-7; TBPH).
Principles of method if other than guideline:
A QSAR Toolbox 3.3.5 query was conducted to evaluate data and the profile of bis(2-ethylhexyl) tetrabromophthalate (CAS No 26040-51-7; TBPH).
GLP compliance:
no
Type of method:
other: QSAR
Endpoint addressed:
not applicable
Species:
other: not applicable - QSAR Toolbox 3.3.5 request
Strain:
other: not applicable - QSAR Toolbox 3.3.5 request
Route of administration:
other: not applicable - QSAR Toolbox 3.3.5 request
Vehicle:
other: not applicable - QSAR Toolbox 3.3.5 request
Duration of treatment / exposure:
not applicable - QSAR Toolbox 3.3.5 request
Frequency of treatment:
not applicable - QSAR Toolbox 3.3.5 request
Post exposure period:
not applicable - QSAR Toolbox 3.3.5 request
No. of animals per sex per dose:
not applicable - QSAR Toolbox 3.3.5 request
Details on results:
TBPH has a molecular weight of 706.15 g/mol.

-> According to the QSAR Toolbox 3.3.5, TBPH is a non-binder to the estrogen receptor based on a MW > 500.

Substances with MW > 500 are Non-ER binder due to high molecular weight:
Estrogen receptor (ER) binding is a molecular initiating event much like protein binding (1) that may lead to a series of adverse outcomes, which are typically linked to reproductive and development hazards. It is an endpoint where several comprehensive databases exist, which has lead to the development of several approaches for using (Q)SARs to predict ER-binding and possible subsequent endocrine disruption (2). Popular among these are the “four phase” assessment that includes Comparative Molecular Field Analysis (CoMFA) (3) and the Common Reactivity Pattern Approach (COREPA) (4).

Since the ER-binding is a receptor mediated event, particular organic functional groups, size and shape are critical to binding potency.

Chemicals that are too large cannot bind to the receptor regardless of structure or shape. While chemicals with a Molecular Weight of greater than 1000 are reported to be too large to bind to the receptor (2, 3) a review of the ER-binding database (ERBA OASIS) within the QSAR Toolbox reveals that no chemical with a molecular weight greater than 500 has been shown to bind to the estrogen receptor (cited from the QSAR Toolbox 3.3.5).
Executive summary:

TBPH has a molecular weight of 706.15 g/mol.

According to the QSAR Toolbox 3.3.5, TBPH is a non-binder to the estrogen receptor based on a MW > 500.

Substances with MW > 500 are Non-ER binder due to high molecular weight: Estrogen receptor (ER) binding is a molecular initiating event much like protein binding (1) that may lead to a series of adverse outcomes, which are typically linked to reproductive and development hazards. It is an endpoint where several comprehensive databases exist, which has lead to the development of several approaches for using (Q)SARs to predict ER-binding and possible subsequent endocrine disruption (2). Popular among these are the “four phase” assessment that includes Comparative Molecular Field Analysis (CoMFA) (3) and the Common Reactivity Pattern Approach (COREPA) (4).

Since the ER-binding is a receptor mediated event, particular organic functional groups, size and shape are critical to binding potency. Chemicals that are too large cannot bind to the receptor regardless of structure or shape. While chemicals with a Molecular Weight of greater than 1000 are reported to be too large to bind to the receptor (2, 3) a review of the ER-binding database (ERBA OASIS) within the QSAR Toolbox reveals that no chemical with a molecular weight greater than 500 has been shown to bind to the estrogen receptor (cited from the QSAR Toolbox 3.3.5).

(1) Schultz, T.W., Carlson. R.E., Cronin, M.T.D., Hermens, J.L.M., Johnson, R., O'Brien, P.J., Roberts, D.W., Siraki, A., Wallace, K.D. and Veith, G.D. 2006. A conceptual framework for predicting toxicity of reactive chemicals: Models for soft electrophilicity. SAR QSAR in Environmental Research 17: 413-428.

(2) Cronin, M.T.D. and Worth A.P. 2008. (Q)SARs for predicting effects relating to reproductive toxicity. QSAR & Combinational Science 27: 91-100.

3) Tong, W., Fang, W.D., Hong, H., Xie, Q., Perkins, R. and Sheehan, D.M. 2004. Receptor-mediated toxicity: QSARs for estrogen receptor binding and priority setting of potential estrogenic endocrine disruptors. In: Cronin, M.T.D. and Livingstone D.J. (Eds) Predicting Chemical Toxicity and Fate. CRC Press Boca Raton FL pp.285-314.

(4) Schmieder, P.K., Ankley, G., Mekenyan, O., Walker, J.D., and Bradbury S. 2003. Environmental Toxicology and Chemistry 22: 1844-1854.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Special investigation
Principles of method if other than guideline:
The metabolism of TBPH (bis(2-ethylhexyl) tetrabromophthalate) was examined in adult female Sprague-Dawley rats.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
basic toxicokinetics
Specific details on test material used for the study:
Technical grade Uniplex FRP-45 (>95 % BEH-TEBP) was obtained from Unitex Chemical Corporation (Greensboro, NC).
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
single application
Frequency of treatment:
single application
Post exposure period:
Approx. 48 h after administration, the rats were euthanized and serum collected.
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Nine adult female Sprague-Dawley rats were administered by gavage a dose of 500 mg/kg bw of technical grade Uniplex FRP-45 (> 95% TBPH) in corn oil. Three other rats used as control received only corn oil.
Control animals:
yes, concurrent vehicle
Details on results:
No TBPH or oxidative metabolites similar to those formed by the DEHP were reported by the authors in urine or serum. TBPA (tetrabromo phthalic acid) was identified as an in vivo urinary and serum metabolite (mean urinary levels ca. 0.5 mg/L and mean serum levels ca. 0.05 mg/L). Additionally, 2,3,4,5-tetrabromobenzoic acid (TBBA) (mean urinary levels ca. 45.6 mg/L and mean serum levels ca. 1.2 mg/mL), a known metabolite of 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) at concentrations much higher than TBPA was detected, even thought TBPH was the main component of Uniplex FRP-45 and TBB was only a minor constituent.

The authors of the study “hypothesized that because of its relatively low solubility and high molecular weight, BEH-TEBP [=TBPH] may excrete preferentially unchanged in feces".

Executive summary:

The metabolism of TBPH was also examined. Nine adult female Sprague-Dawley rats were administered by gavage a dose of 500 mg/kg bw of technical grade Uniplex FRP-45 (> 95% TBPH) in corn oil. Three other rats used as control received only corn oil. For each of the nine rats, 24-h urine samples were collected 1 day before and 24 h after administration of corn oil with and without Uniplex FRP-45. Approx. 48 h after administration, the rats were euthanized and serum was collected. Feces were not collected for this experiment.

No TBPH or oxidative metabolites similar to those formed by the DEHP were reported by the authors in urine or serum. TBPA was identified as an in vivo urinary and serum metabolite (mean urinary levels ca. 0.5 mg/L and mean serum levels ca. 0.05 mg/L). Additionally, 2,3,4,5-tetrabromobenzoic acid (TBBA) (mean urinary levels ca. 45.6 mg/L and mean serum levels ca. 1.2 mg/mL), a known metabolite of 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) at concentrations much higher than TBPA was detected, even thought TBPH was the main component of Uniplex FRP-45 and TBB was only a minor constituent.

The authors of the study “hypothesized that because of its relatively low solubility and high molecular weight, BEH-TEBP [= TBPH] may excrete preferentially unchanged in feces. Regrettably, we did not collect feces for this experiment”.

Endpoint:
endocrine system modulation
Type of information:
other: Study with the monoester metabolite of bis(2-ethylhexyl) tetrabromophthalate (TBPH)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Special investigation
Principles of method if other than guideline:
The rodent thyroid, liver, and fetal testis toxicity of the Monoester Metabolite of TBPH, was investigated.
GLP compliance:
no
Type of method:
other: in vivo + in vitro
Specific details on test material used for the study:
TBPH and TBMEHP were synthesized by AsisChem (Watertown, MA)
Species:
rat
Strain:
other: Fisher
Sex:
female
Route of administration:
oral: gavage
Vehicle:
other: corn oil and 5% ethanol
Duration of treatment / exposure:
Rat dams were dosed once per day on gestational days (GDs) 18 and 19
Frequency of treatment:
Rat dams were dosed once per day on gestational days (GDs) 18 and 19
Post exposure period:
Six hours after the final dose, dams were euthanized by isoflurane overdose and cervical dislocation.
No. of animals per sex per dose:
Three treatment groups:
corn oil/ethanol control (n = 10),
200 mg TBMEHP/kg (low dose; n = 9), and
500 mg TBMEHP/kg (high dose; n = 10).
Control animals:
yes, concurrent vehicle
Details on results:
In pregnant Fischer rats gavaged daily for 2 days (GD18 and GD19) with TBMEHP, no significant differences in liver, kidney, adrenal gland, and ovary weights compared with vehicle controls was found. In dams that received the high TBMEHP dose (500 mg/kg), the liver enzyme alkaline phosphatase was significantly decreased but alanine transaminase was significantly increased. Also in the high-dose group, blood urea nitrogen levels were significantly higher and calcium levels were significantly lower. Cholesterol levels decreased significantly in a dose-dependent manner. Serum T3 was significantly reduced in a dose-dependent-manner, but no effect on serum T4 was observed.

Given the effects on dam livers, kidneys, and thyroids indicated by the serum clinical chemistries, these organs were evaluated histopathologically. In TBMEHP-exposed dams, no abnormalities were observed in the kidneys or thyroids, whereas effects were observed in the livers. Qualitatively, liver sections from TBMEHP-exposed dams showed an increase in hepatocytes with mitotic spindles indicating proliferation, and an increase in hepatocytes with dense hypereosinophilic cytoplasm and condensed, fragmented nuclei characteristic of apoptosis. These qualitative impressions were confirmed by quantifying the number of cells in mitosis across groups, with a significant increase in mitotic activity observed in high-dose dams.

Cross-sections of fetal testes were scored for the number of MNGs; in high-dose fetal testes we found a significantly increased number of MNGs per cord area. After ex vivo incubation of fetal testes in media, no significant changes in testosterone production in treated animals compared with controls (control, 1.47 ± 0.06 μg/dL; low dose, 1.36 ± 0.11 μg/dL; high dose, 1.28 ± 0.05 μg/dL) were observed.

Deiodinase activity. Co-incubation of TBMEHP and T4 with rat hepatic microsomes resulted in a dose-dependent decrease in deiodinase activity.

The mechanistic experiments revealed that TBMEHP activates both PPARα- and PPARγ-mediated gene transcription and is capable of stimulating PPARγ-mediated adipocyte differentiation.

Altogether, TBMEHP occurs as a product of mammalian esterase activity. TBMEHP was toxicologically assessed by exposing pregnant rat dams to 200 or 500 mg TBMEHP/kg or to vehicle. After 2 days of exposure (GD18 and GD19), serum liver enzyme levels of the dams were altered, and histopathological analysis of dam livers showed hepatocyte apoptosis and proliferation. Serum T3 levels were significantly decreased in a dose-dependent manner and that TBMEHP inhibited deiodinase activity. Testes isolated from the fetuses of exposed dams showed induction of MNGs in the high-dose group without significant effects on testosterone production. In in vitro mechanistic studies using murine FAO and NIH 3T3 L1 cells, TBMEHP as a PPARα and PPARγ agonist was identified.
-> With TBMEHP similar effects were seen as compared with DEHP.

DEHP is a known peroxisome proliferator and male reproductive toxicant in rodents. DEHP is metabolized by esterases to MEHP, its toxicologically active monoester metabolite. DEHP induces hepatotoxicity in rodents, most likely as a result of MEHP-induced activation of PPARα. The developing male reproductive system in rats is highly sensitive to the effects of these phthalates, which decrease fetal male testosterone levels. The active phthalates disrupt steroidogenesis in fetal rat Leydig cells, and this antiandrogenic effect impairs the normal development of the male reproductive tract. The active phthalates also alter fetal testis seminiferous cords, an effect manifested by the induction of MNGs.
-> However, in-vitro experiments indicate that the hydrolysis of DEPH is approx. 100 times faster than the hydrolysis of TBPH.
-> Additionally, the bioavailability of DEHP is much higher as the bioavailability of TBPH.

DEHP (water solubility = ca. 0.003 mg/L, Log Pow = 7 to 8)
TBPH (water solubility < 0.05 µg/L, Log Pow = 10.2)

Therefore, based on the much lower bioavailability and the much lower hydrolysis rate of TBPH compared with DEHP it is expected that the results of TBMEHP are not applicable to TBPH. This is in contrast to MEHP the toxicologically active monoester metabolite of DEHP, were the hydrolysis rate of the parent compound is 100 times faster compared with TBPH.
This interpretation based on metabolism/kinetic data is confirmed by the available 28 day study on TBPH and DEHP (see IUCLID section 7.5)
Executive summary:

The rodent thyroid, liver, and fetal testis toxicity of the Monoester Metabolite of TBPH, was investigated (12):

In vivo rat experiments. Timed-pregnant Fischer rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were assigned by randomized weight to three treatment groups: corn oil/ethanol control (n = 10), 200 mg TBMEHP/kg (low dose; n = 9), and 500 mg TBMEHP/kg (high dose; n = 10). TBMEHP, which is a water-insoluble solid at room temperature, was dissolved in a solution of corn oil and 5% ethanol. Dosing was by gavage (2 mL/kg).

Rat dams were dosed once per day on gestational days (GDs) 18 and 19. Six hours after the final dose, dams were euthanized by isoflurane overdose and cervical dislocation. Blood was collected by cardiac puncture, and serum was isolated by centrifugation and then frozen at –80°C until further use. Organs were harvested and weighed, then fixed in 10% neutral buffered formalin. Fetuses were euthanized by decapitation, and sex was identified by internal dissection. Within each litter, both testes of male pups were numbered upon isolation.

The effects of in utero exposure on the fetal testes was examined. Formalin-fixed fetal testes were embedded in glycol methacrylate, sectioned (5 µm), and stained with H&E. The sections were scanned using an Aperio CS ScanScope, and the digitized images were used to measure seminiferous cord area. The testis slides were then scored under a microscope for the presence of multinucleated germ cells (MNGs); the number of MNGs per testis was normalized by semini-ferous cord area and then pooled by litter.

Testosterone production in the fetal testis by incubating single fetal testes in M 199 media at 37°C for 3 hr. The media supernatant was collected in aliquots and analyzed for testosterone by immunoassay.

Deiodinase inhibition. The effects of TBMEHP on deiodinase activity using a competitive substrate assay were investigated.

TBMEHP was also assessed for peroxisome proliferator-activated receptor (PPAR) α and γ activation using murine FAO cells and NIH 3T3 L1 cells.

Results: In pregnant Fischer rats gavaged daily for 2 days (GD18 and GD19) with TBMEHP, no significant differences in liver, kidney, adrenal gland, and ovary weights compared with vehicle controls was found. In dams that received the high TBMEHP dose (500 mg/kg), the liver enzyme alkaline phosphatase was significantly decreased but alanine transaminase was significantly increased. Also in the high-dose group, blood urea nitrogen levels were significantly higher and calcium levels were significantly lower. Cholesterol levels decreased significantly in a dose-dependent manner. Serum T3 was significantly reduced in a dose-dependent-manner, but no effect on serum T4 was observed.

Given the effects on dam livers, kidneys, and thyroids indicated by the serum clinical chemistries, these organs were evaluated histopathologically. In TBMEHP-exposed dams, no abnormalities were observed in the kidneys or thyroids, whereas effects were observed in the livers. Qualitatively, liver sections from TBMEHP-exposed dams showed an increase in hepatocytes with mitotic spindles indicating proliferation, and an increase in hepatocytes with dense hypereosinophilic cytoplasm and condensed, fragmented nuclei characteristic of apoptosis. These qualitative impressions were confirmed by quantifying the number of cells in mitosis across groups, with a significant increase in mitotic activity observed in high-dose dams.

Cross-sections of fetal testes were scored for the number of MNGs; in high-dose fetal testes we found a significantly increased number of MNGs per cord area. After ex vivo incubation of fetal testes in media, no significant changes in testosterone production in treated animals compared with controls (control, 1.47 ± 0.06 μg/dL; low dose, 1.36 ± 0.11 μg/dL; high dose, 1.28 ± 0.05 μg/dL) were observed.

Deiodinase activity. Co-incubation of TBMEHP and T4 with rat hepatic microsomes resulted in a dose-dependent decrease in deiodinase activity.

The mechanistic experiments revealed that TBMEHP activates both PPARα- and PPARγ-mediated gene transcription and is capable of stimulating PPARγ-mediated adipocyte differentiation.

Altogether, TBMEHP occurs as a product of mammalian esterase activity. TBMEHP was toxicologically assessed by exposing pregnant rat dams to 200 or 500 mg TBMEHP/kg or to vehicle. After 2 days of exposure (GD18 and GD19), serum liver enzyme levels of the dams were altered, and histopathological analysis of dam livers showed hepatocyte apoptosis and proliferation. Serum T3 levels were significantly decreased in a dose-dependent manner and that TBMEHP inhibited deiodinase activity. Testes isolated from the fetuses of exposed dams showed induction of MNGs in the high-dose group without significant effects on testosterone production. In in vitro mechanistic studies using murine FAO and NIH 3T3 L1 cells, TBMEHP as a PPARα and PPARγ agonist was identified.

 With TBMEHP similar effects were seen as compared with DEHP.

DEHP is a known peroxisome proliferator and male reproductive toxicant in rodents. DEHP is metabolized by esterases to MEHP, its toxicologically active monoester metabolite. DEHP induces hepatotoxicity in rodents, most likely as a result of MEHP-induced activation of PPARα. The developing male reproductive system in rats is highly sensitive to the effects of these phthalates, which decrease fetal male testosterone levels. The active phthalates disrupt steroidogenesis in fetal rat Leydig cells, and this antiandrogenic effect impairs the normal development of the male reproductive tract. The active phthalates also alter fetal testis seminiferous cords, an effect manifested by the induction of MNGs.

 However, in-vitro experiments indicate that the hydrolysis of DEPH is approx. 100 times faster than the hydrolysis of TBPH.

 Additionally, the bioavailability of DEHP is much higher as the bioavailability of TBPH.

DEHP (water solubility = ca. 0.003 mg/L, Log Pow = 7 to 8)

TBPH (water solubility < 0.05 µg/L, LogPow = 10.2)

Therefore, based on the much lower bioavailability and the much lower hydrolysis rate of TBPH compared with DEHP it is expected that the results of TBMEHP are not applicable to TBPH. This is in contrast to MEHP the toxicologically active monoester metabolite of DEHP, were the hydrolysis rate of the parent compound is 100 time faster compared with TBPH. This interpretation based on metabolism/kinetic data is confirmed by the available 28 day study on TBPH and DEHP (see IUCLID section 7.5).

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Principles of method if other than guideline:
Proliferation of the estrogen receptor α-positive human breast cancer cell line, MCF-7, has been routinely used as a measure of estrogenicity.
GLP compliance:
not specified
Type of method:
in vitro
Details on study design:
The MCF-7 flow cytometry cell proliferation assay has been described previously (Vanparys et al., 2006). Briefly, 50,000 cells were seeded into 6-well plates and allowed to attach. Following cell-cycle synchronisation in exposure medium for 48 h, compounds were added and cells treated for 24 h. Depending on endpoint measured (anti-estrogenicity or estrogenicity) cells were either co-treated with orwithout 17β-estradiol (E2) (100 pM). The proliferative potential of a FR compound was indicated by the number of cells in the DNA synthesis stage of the cell cycle, the S-phase. Results are presented as the mean value of three independent experiments.
Details on results:
TBPH was tested in the estrogen receptor α-positive human breast cancer cell line MCF-7 for the induction of cell proliferation, a screening assay for potential estrogenic effects. The concentrations tested were reported to exert no relevant cytotoxicity. It was shown in this study that TBPH at concentrations of up to 100 µM did neither induce nor inhibit cell proliferation. Thus, no estrogenic and no anti-estrogenic activity was seen for TBPH.
Executive summary:

TBPH was tested in the estrogen receptor α-positive human breast cancer cell line MCF-7 proliferation assay (Krivoshiev et al., 2016), a screening test for potential estrogenic effects. The concentrations tested were reported to exert no relevant cytotoxicity. It was shown in this study that TBPH at concentrations of up to 100 µM did neither induce nor inhibit cell proliferation. Thus, no estrogenic and no anti-estrogenic activity was seen for TBPH.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
no guideline followed
Type of method:
in vitro
Endpoint addressed:
other: endocrine modulation

The results showed no estogenic and androgenic activity of TBB (2-ethylhexyl-2,3,4,5-tetrabromobenzoate) and TBPH in the yeast reporter assay (YES/YAS modified version). Antagonistic effects were observed on both receptors at 25 µM and at 48 µM TBPH for ER and AR, respectively. In addition to the parent compounds Fic et al. also tested potential hydrolysis metabolites, i.e. TBMEPH (2-ethylhexyltetrabromobenzoate ester) for the parent compound TBPH. The TBMEPH metabolite with an IC50 value of 265 nM was the most active among all tested compounds in the ER antagonist YES assay. The authors conclude that metabolism can enhance the anti-estrogenic and anti-androgenic activities of those two novel brominated flame retardants.

Executive summary:

The results showed no estogenic and androgenic activity of TBB (2-ethylhexyl-2,3,4,5-tetrabromobenzoate) and TBPH in the yeast reporter assay (YES/YAS modified version). Antagonistic effects were observed on both receptors at 25 µM and at 48 µM TBPH for ER and AR, respectively. In addition to the parent compounds Fic et al. also tested potential hydrolysis metabolites, i.e. TBMEPH (2-ethylhexyltetrabromobenzoate ester) for the parent compound TBPH. The TBMEPH metabolite with an IC50 value of 265 nM was the most active among all tested compounds in the ER antagonist YES assay. The authors conclude that metabolism can enhance the anti-estrogenic and anti-androgenic activities of those two novel brominated flame retardants.

Endpoint:
specific investigations: other studies
Type of information:
other: collection of data
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Principles of method if other than guideline:
collection of data
Endpoint addressed:
other: toxicity in general
Specific details on test material used for the study:
TBPH (called BEH-TEBP in this paper)

In a recent publication of Bajard et al. (2019) 62 flame retardants including TBPH (called BEH-TEBP in the paper) were prioritized regarding their hazards, based on their current regulatory status, in vivo toxicological data collected from the literature, ToxCast data and the AOP-wiki. As relevant ToxCast data for TBPH activation of the glucocorticoid receptor in the assay NVS_NR_hGR with an AC50 of 4.96 µM is given. The origin of the in vivo data (9 publications) is not specified. However, it is stated that for TBPH ‘the evidence was mostly based on studies of a chemical mixture (such as FireMaster 550), which contains a number of other FRs’. The toxicological concern was determined with low/some.

Endpoint:
biochemical or cellular interactions
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
androstane receptor binding test; no guideline followed
Type of method:
in vitro
Endpoint addressed:
other: receptor binding test

The study of Zhang H et al. (2015) investigated the human constitutive androstane receptor  (CAR) binding activities of 23 phthalate esters and 10 phthalate monoesters using a fast and sensitive human CAR yeast two-hybrid assay. CAR is a member of the nuclear receptor superfamily, and is a key regulator of xenobiotic and endobiotic metabolism. The 10% relative effective concentration (REC10) of TBMEHP was 0.66 μM. No significant activation of human CAR was observed after incubation with the parent compound TBHP, even at the highest concentration tested (100 μM). A molecular docking method was performed to simulate the interaction modes between phthalates and human CAR, and active phthalates were found to lie at almost the same site in the human CAR pocket. The docking results suggest that the strong binding of phthalates to human CAR arises primarily from hydrophobic interactions, π−π interactions, and steric effects and that weak hydrogen bonds and weak halogen bonds greatly contribute to the high binding activity of TBMEHP.

Endpoint:
cytotoxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
in vitro test with human vascular endothelial cells; no guideline followed
Type of method:
in vitro
Endpoint addressed:
other: in vitro effects on human vascular cells

The effects of TBPH and TBMEHP on human vascular endothelial cells (HUVECs) were investigated by Xiang et al. (2017). A concentration-dependent inhibition on HUVECs' viability and growth was observed for TBMEHP but not for TBPH. TBMEHP induced a marked G0/G1 cell cycle arrest and robust cell apoptosis at 1 μg/mL by inducing expression of p53, GADD45α and cyclin dependent kinase (CDK) inhibitors (p21and p27) while suppressing the expression of cyclin D1, CDK2, CDK6, and Bcl-2. Unlike TBMEHP, TBPH caused early apoptosis after G2/M phase arrest only at 10 μg/mL via upregulation of  21 and down-regulation of CDK2 and CDK4. TBMEHP decreased mitochondrial membrane potential and increased caspase-3 activity at 1 μg/mL, suggesting that activation of p53 and mitochondrial pathway were involved in the cell apoptosis. The data showed that TBPH and TBMEHP induced different cell cycle arrest and apoptosis through different molecular mechanisms with much higher toxicity for TBMEHP.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: special investigation
Principles of method if other than guideline:
specific in vitro investigation
Endpoint addressed:
other: specific investigation
Details on test animals or test system and environmental conditions:
in vitro investigation

TBPH was investigated in vitro for peroxisome proliferator-activated receptor gamma (PPARγ) in Chinese Hamster Ovary (CHO) cell line based human nuclear receptor luciferase reporter assay systems (Belcher et al., 2014). No activity was observed for TBPH (concentrations tested ranged from 10-12 to 10-4 M).

Executive summary:

TBPH was investigated in vitro for peroxisome proliferator-activated receptor gamma (PPARγ) in Chinese Hamster Ovary (CHO) cell line based human nuclear receptor luciferase reporter assay systems (Belcher et al., 2014). No activity was observed for TBPH (concentrations tested ranged from 10-12 to 10-4 M).

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis; no guideline followed
Endpoint addressed:
other: glucocorticoid receptor mediated adipogenesis in vitro

A new in vitro approach for investigation of effects on glucocorticoid receptor mediated adipogenesis is described by Hartman et al. (2018). The primary human adipose stem cell (hASC) assay is used to evaluate the effect of environmental chemicals on PPARγ-dependent adipogenesis. The assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes.19 chemicals with suspected GR-dependent activity were tested. TBPH was the only chemical identified as an agonist in ToxCast that did not demonstrate GR activity in the adipogenesis assay.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
PPARα and PPARγ agonist and antagonist activities and agonist activity on pregnane X receptor; no guideline followed
Endpoint addressed:
other: endocrine modulation

TBPH and its metabolite TBMEPH were evaluated for PPARα and PPARγ agonist and antagonist activities by Skledar et al. (2016). None of these chemicals showed marked activity. Concentrations of 10 and 50 µM were tested. TBPH and its metabolite TBMEPH were also investigated for their agonist activity on pregnane X receptor (PXR) by Skledar et al. (2016). PXR is a member of the nuclear receptor superfamily that is involved in xenobiotic detoxification through the regulation of drug metabolising enzymes and transporters, such as cytochromes P450 (CYPs) and the ATP-binding cassette (ABC) drug transporters. EC50 values of 5.5 µM for TBPH and 2.0 µM for its metabolite were reported. Additionally, TBPH showed antagonist activity on PXR (IC50, 13.9 µM). Moreover, there was significant up-regulation of CYP3A4 expression via PXR activation for TBPH and its metabolite.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
reporter gene assays in vitro; no guideline followed
Type of method:
in vitro
Endpoint addressed:
other: endocrine modulation
Details on study design:
influences on glucocorticoid receptor (GR), thyroid hormone receptor (TR) and androgen receptor (AR) were determined and compared using luciferase expression

In a study of Klopcic et al. (2016) two novel brominated flame retardants, TBB (2-ethylhexyl-2,3,4,5-tetrabromobenzoate) and TBPH and their metabolites TBBA and TBMEPH were investigated in three different reporter gene assays. The influences on glucocorticoid receptor (GR), thyroid hormone receptor (TR) and androgen receptor (AR) were determined and compared using luciferase expression. The highest non cytotoxic concentrations were 50 µM and 75 µM for TBPH and TBMEPH, respectively, in MDA-kb2 cell cultures for the AR and GR reporter gene assay, and 25 µM and 100 µM for TBPH and TBMEPH, respectively, in GH3.TRE-LUC cell cultures for the TR reporter gene and resazurin assay. The study revealed antiandrogenic effects (IC50 of 0.1 and 1.3 µM) and anti-thyroid hormonal effects (IC50 of 0.1 and 32.2 µM) of TBPH its metabolite TBMEPH, but no agonistic activities. The parent compounds TBB and TBPH also showed anti-glucocorticoid effects as well as competition for binding to the GR.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: explorative study
Principles of method if other than guideline:
inhibitory effects of five novel brominated flame retardants on thyroid hormone deiodinase (DIO) and sulfotransferase (SULT) activity were investigated using human in vitro liver microsomal and cytosolic bioassays; no Guidline followed
Endpoint addressed:
other: endocrine modulation
Details on study design:
Enzymatic activity was measured by incubating active human liver subcellular fractions with thyroid hormones (T4 and rT3 separately) and measuring changes in thyroid hormone (T4, T3, rT3, and 3,3′-T2) concentrations in the presence of the flame retardants. IC50 values for outer and inner ring deiodination (ORD and IRD) are given.

The inhibitory effects of five novel brominated flame retardants, including bis(2-ethylhexyl)-tetrabromophthalate (TBPH, called BEH-TEBP in this study), on thyroid hormone deiodinase (DIO) and sulfotransferase (SULT) activity were investigated using human in vitro liver microsomal and cytosolic bioassays (Smythe et al., 2017). Enzymatic activity was measured by incubating active human liver subcellular fractions with thyroid hormones (T4 and rT3 separately) and measuring changes in thyroid hormone (T4, T3, rT3, and 3,3′-T2) concentrations in the presence of the flame retardants. IC50 values for outer and inner ring deiodination (ORD and IRD) are given. TBPH concentrations of 3.75 nM to 3.75 µM were tested. No effects on T4 conversion to its deiodinated isoforms were seen, with the exception of TBPH in 3,3′-T2 and T3 formation at the highest concentration tested (3.75 μM, p <0.05). However, the authors admit that 3.75 μM likely far exceeds environmentally relevant levels for TBPH. No significant SULT inhibition was recorded for any of the tested flame retardants. The authors therefore conclude no significant impact on DIO and SULT for TBPH.

Description of key information

A comprehensive literature search was conducted and toxicological databases queried for potential endocrine effects of bis(2-ethylhexyl) tetrabromophthalate (CAS No 26040-51-7; TBPH).  Altogether, there is no consistent indication for a potential endocrine activity. The assumption that TBPH fulfills the criteria as potential endocrine disruptor based on structural similarity to DEHP is not confirmed by the available experimental data.

Additional information

Assessment of potential endocrine activity (toxicity/human health) - summary and conclusion

A comprehensive literature search was conducted to identify data on potential endocrine modulating properties of TBPH. The available data was assigned to the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters levels 1 to 5.

TBPH can be described as tetrabrominated DEHP. Whereas DEHP is a known endocrine active substance TBPH falls into disrepute due to its molecular similarity with DEHP. This assessment shows that the bromination in TBPH results in a substantial different behaviour compared to DEHP with respect to metabolism, toxicity, reproduction toxicity and binding affinities to endocrine receptors.

It is postulated that DEHP and its brominated counterpart TBPH are metabolized in a similar manner to the monoester metabolites MEHP and TBMEHP, respectively. This metabolic pathway is proven for DEHP, and MEHP is identified as the responsible metabolite for the toxicity of DEHP. For TBPH this metabolism is also proven, however, only in vitro in the presence of porcine esterases. No formation of TBMEHP was identified for TBPH under realistic conditions, neither with human hepatocellular fractions nor in vivo in rats or in mice under realistic exposure conditions.

TBPH is determined as ‘non-binder to the estrogen receptor’ by the OECD Toolbox Version 3.3.5 based on a MW of > 500 g/mol.

TBPH showed mainly negative and few positive results in some of the in vitro screening assays for anti/androgenic and anti/estrogenic activity, but no conclusive picture is given. The activities were seen in general only at concentrations in the micromolar range and at magnitudes higher concentrations than TBPH detected in human body fluids, and are thus not relevant for the in vivo situation. Based on the available information there is no indication of a specific endocrine activity in vitro.

This conclusion is in line with the available animal studies.

In animal studies TBPH is well tolerated. The overall NOEL for repeated dose toxicity is determined with 1000 mg/kg bw/day.

There were no gross or microscopic findings to the reproductive system and to reproductive parameters in repeated oral dose toxicity studies (28- and 90-day studies) and a developmental toxicity study in rats that would point to an endocrine activity of TBPH. This is in contrast to the effects seen for the unbrominated DEHP, used as positive control in the 28-day oral toxicity study with TBPH. For DEHP strong general toxicity and severe testes effects in rats at daily doses lower than that of TBPH were recorded.

In conclusion, there is no reliable evidence of a specific and biologically relevant endocrine activity of TBPH throughout all OECD CF level studies, i.e. in vitro assays and experimental animal studies.

An endocrine disruptor is defined by WHO as an ‘exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub) populations.’

Based on the information available it can be concluded that TBPH does not meet the WHO definition of an endocrine disruptor with respect to human health.

The complete assessment report is attached as pdf to the endpoint summary of IUCLID chapter 7.9.4 and to IUCLID chapter 13.2.