1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromopropoxy)benzene]

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: Publication

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study. Not performed according to GLP and standard testing guidelines.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Materials and methods

Objective of study:

other: The primary objectives were to measure the absorption, distribution, metabolism and elimination of TBBA-DBPE following oral or intravenous(IV) administration to male Fischer-344 (F-344) rats.

Principles of method if other than guideline:

Blood kinetics following oral and intravenous administration of TBBA-DBPE
[14C] TBBA-DBPE (20mg/kg) was administered IV through JVC (50Ci/kg, 1 mL/kg) or by oral gavage (200Ci/kg, 4 mL/kg) in a solution of CremophorEL; saline; DMSO, 30;65;5 (v/v/v).
Following IV administration, the cannula was flushed with whole blood (100L) and normal saline (1mL/kg). Blood samples (300L) were collected via the JVC at 0.125, 0.25, 0.5, 1, 3, 6, 12, 24, 36, 48 (PO and IV), 72, 96 hours (IV) into heparinized syringes. After each blood collection (300L) of normal saline was infused to replace blood volume. Blood from these samples were solubilized and radioactivity was determined by liquid scintillation counting (300L) (LSC). Additionally, blood samples (200L) were analyzed by HPLC-UV/Vis-radio to determine the nature of [14C] equivalents present in blood.

Tissue distribution and excretion studies
Following administration of [14C] TBBA-DBPE (20mg/kg, 50Ci/kg) by IV (1 mL/kg) or oral route (2-4mL/kg), the animals were placed in Nalgene metabolism cages for collection of feces and urine. Urine was collected at 6, 12, 24, 36, 48, 72, and 96 hours. Feces were collected at 12, 24, 36, 48, 72, and 96 hours. Cages were rinsed with methanol following collection of urine. Cage rinse were collected and analyzed separately from urine samples. At the end of each study, animals were euthanized by CO2 inhalation and subjected to necropsy. Tissues (brain, heart, lung, kidneys, liver, spleen, stomach, stomach content, intestine, intestinal content, cecum, cecum content, testes, muscle, fat and skin) were immediately placed on ice at necropsy and then stored at -20o until analyzed. Feces were solubilized using Soulene-350, while tissues, blood and bile were solubilized using Solvable. Estimated % of body weight composition used to calculate % of dose were: 11% adipose, 9% bold, 50% muscle, and 16% skin. Radioactivity in urine and cage rinse was determined by direct LSC analysis.

Biliary excretion study
BDC F-344 rats were dosed with [14C] TBBA-DBPE by oral gavage (20mg/kg, 50Ci/kg, 4mL/kg) in a solution of CremophorEL; saline; DMSO, 30;65;5 (v/v/v). Immediately after dosing, animals were returned to Nalgene metabolism cages that had been modified using a Metamount plastic swivel and swivel mount (Instech Laboratories, Inc.; Plymouth Meeting, PA). BDC cannulae were threaded through the swivel to permit continuous bile collection without restraint for the duration of the study. The bile was collected for 24h at 2h intervals. At the termination of these experiments necropsies were performed as described in the previous section.

Toxicokinetics analysis
The blood concentration-time data following IV and oral administration were analyzed using modeling program (WinNonlin, Pharsight Corp., Mountain View, CA). A two compartment model that assumed first order elimination was found to be the best model to fit the data sets. The model was used to calculate value the half-life of distribution (t1/2), terminal half-life for elimination (t1/2), area under the blood concentration-time curve from time zero to infinity (AUC), volume of distribution at steady state (Vss), apparent clearance from blood (CLb), calculated maximum concentration of TBBA-DBPE in the blood (Cmax) and maximum oral bioavailability (f).
In vitro techniques
Microsomal protein (MIC) was isolated from male F-344 rat livers and quantified. [14C] TBBA-DBPE (10-100M, 4.8Ci/mL) was incubated with 1, 2, or 5mg/mL MIC in HEPES buffer containing a recycling NADP+ system. Aliquots were removed at various times (0-4h) and 2 equivalents of acetonitrile were added to terminate enzyme activity. Samples were vortexed, sonicated for 5 minutes, centrifuged at 13,000RPM for 2 minutes and the supernatant analyzed by HPLC-UV/Vis-radio. De-ethylation of 7-ethoxycoumarin to 7-hydroxycumarin was used as a positive indicator of microsomal P450 activity.
Hepatocytes (HC) in were isolated from male F-344 rats. [14C] TBBA-DBPE (50 and 100M, 190Ci/L) was incubated with HC in suspension (1x106 cells/ mL in 6 well cell culture plates), with shaking (70RPM) at 37oC, or in plated monolayers. Aliquots of media were removed at various times (0-4h: HC suspension; 0-24h: HC monolayers), treated, and analyzed. Conjugation of [14C] TBBA-DBPE and [14C] BPA to [14C] TBBA and [14C] BPA-glucuronide, respectively, was used to confirm metabolic activity.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromopropoxy)benzene]

EC Number:

244-617-5

EC Name:

1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromopropoxy)benzene]

Cas Number:

21850-44-2

Molecular formula:

C21H20Br8O2

IUPAC Name:

1,1'-propane-2,2-diylbis[3,5-dibromo-4-(2,3-dibromopropoxy)benzene]

Details on test material:

TBBA-DBPE [14C], phenol ring labelled (0.97mCi/mL in DMSO), TBBA-[14C], phenol ring labelled were obtained from Perkin-Elmer Life and Analytical Science (Boston, MA). Bispheol phenol A-[14C], phenol ring labelled was obtaind from Moravek Biochemicals (Brea, CA). TBBA-DBPE refernce standars were obtained from TCI America (Portland, Oragon). Unlabelled TBBA and Bispheol A were obtained from Aldrich (St. Louis, MO).
.

Radiolabelling:

yes

Remarks:

TBBA-DBPE [14C], phenol ring labelled.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

Male Fischer 344 rats of 8-9 weeks of age (160-190 gr) were obtained from Harlan Spargue Dawley, Inc. (Indianapolis, IN). Surgical alterations (jugular vein and common bile duct cannulation) were performed at Harlan Spargue Dawley prior to shipment. Conventional animals were acclimatized for 5-7 days. Animals with surgically implanted cannula (JVC or BDC) were acclimatized only for 24 h to ensure the cannula would remain unobstructed over the course of the experiment.
Animals were housed in University Animal Care, an Association for Assessment and Accreditation for Laboratory Animal Care approved animal care facility. They were maintained in a temperature controlled (25o) room, on 12h/12h light/ dark cycle. Upon receipt, animals were placed in hanging steel mesh cages with free access to food and water. Animals were allowed food (Telkad 4% Rat diet 7001, Harlan Telkad, Madison, WI) water ad libitum and except for 12 h prior to single administrations of TBBA-DBPE. Food was returned 2 h post- administration. Animals utilized during the repeated dose study were not fasted.
Animals were placed in Nalgene metabolism cages for acclimation 24h prior to administration of TBBA-DBPE. Animals were maintained in in Nalgene metabolism cages with rat- appropriate parts for the duration of experiments.

Administration / exposure

Route of administration:

other: Oral and intravenous administration

Vehicle:

DMSO

Details on exposure:

See section of study design.

Doses / concentrations

Remarks:

Doses / Concentrations:
The doses used in these in vitro studies were based on the results of published toxicity data (van Esch, 1995*). The dose of 20mg/kg represents a non-toxic dose (approximately 1000-fold lower than published LD50 in rats). This lack of acute toxicity was confirmed following a single IV administration of TBBA-DBPE.
Doses selected for the in vitro assays (100, 50, or 10uM) were designed to assay for metabolic product generation, and were therefore based on stable aqueous concentrations of TBBA-DBPE in the presence of rat hepatocytes or rat liver microsomal protein.
*Van Esch, G.J., 1995. Tetrabromobisphenol A dibromopropylether. In: Environmental Health Criteria 172: Tetrabromobisphenol A and derivatives. International Programme on Chemical Safety. World Health Organization, Finland, pp, 71-77.

Details on study design:

Blood kinetics following oral and intravenous administration of TBBA-DBPE
[14C] TBBA-DBPE (20mg/kg) was administered IV through JVC (50Ci/kg, 1 mL/kg) or by oral gavage (200Ci/kg, 4 mL/kg) in a solution of CremophorEL; saline; DMSO, 30;65;5 (v/v/v).
Following IV administration, the cannula was flushed with whole blood (100L) and normal saline (1mL/kg). Blood samples (300L) were collected via the JVC at 0.125, 0.25, 0.5, 1, 3, 6, 12, 24, 36, 48 (PO and IV), 72, 96 hours (IV) into heparinized syringes. After each blood collection (300L) of normal saline was infused to replace blood volume. Blood from these samples were solubilized and radioactivity was determined by liquid scintillation counting (300L) (LSC). Additionally, blood samples (200L) were analyzed by HPLC-UV/Vis-radio to determine the nature of [14C] equivalents present in blood.

Tissue distribution and excretion studies
Following administration of [14C] TBBA-DBPE (20mg/kg, 50Ci/kg) by IV (1 mL/kg) or oral route (2-4mL/kg), the animals were placed in Nalgene metabolism cages for collection of feces and urine. Urine was collected at 6, 12, 24, 36, 48, 72, and 96 hours. Feces were collected at 12, 24, 36, 48, 72, and 96 hours. Cages were rinsed with methanol following collection of urine. Cage rinse were collected and analyzed separately from urine samples. At the end of each study, animals were euthanized by CO2 inhalation and subjected to necropsy. Tissues (brain, heart, lung, kidneys, liver, spleen, stomach, stomach content, intestine, intestinal content, cecum, cecum content, testes, muscle, fat and skin) were immediately placed on ice at necropsy and then stored at -20o until analyzed. Feces were solubilized using Soulene-350, while tissues, blood and bile were solubilized using Solvable. Estimated % of body weight composition used to calculate % of dose were: 11% adipose, 9% bold, 50% muscle, and 16% skin. Radioactivity in urine and cage rinse was determined by direct LSC analysis.

Biliary excretion study
BDC F-344 rats were dosed with [14C] TBBA-DBPE by oral gavage (20mg/kg, 50Ci/kg, 4mL/kg) in a solution of CremophorEL; saline; DMSO, 30;65;5 (v/v/v). Immediately after dosing, animals were returned to Nalgene metabolism cages that had been modified using a Metamount plastic swivel and swivel mount (Instech Laboratories, Inc.; Plymouth Meeting, PA). BDC cannulae were threaded through the swivel to permit continuous bile collection without restraint for the duration of the study. The bile was collected for 24h at 2h intervals. At the termination of these experiments necropsies were performed as described in the previous section.

Toxicokinetics analysis
The blood concentration-time data following IV and oral administration were analyzed using modeling program (WinNonlin, Pharsight Corp., Mountain View, CA). A two compartment model that assumed first order elimination was found to be the best model to fit the data sets. The model was used to calculate value the half-life of distribution (t1/2), terminal half-life for elimination (t1/2), area under the blood concentration-time curve from time zero to infinity (AUC), volume of distribution at steady state (Vss), apparent clearance from blood (CLb), calculated maximum concentration of TBBA-DBPE in the blood (Cmax) and maximum oral bioavailability (f).
In vitro techniques
Microsomal protein (MIC) was isolated from male F-344 rat livers and quantified. [14C] TBBA-DBPE (10-100M, 4.8Ci/mL) was incubated with 1, 2, or 5mg/mL MIC in HEPES buffer containing a recycling NADP+ system. Aliquots were removed at various times (0-4h) and 2 equivalents of acetonitrile were added to terminate enzyme activity. Samples were vortexed, sonicated for 5 minutes, centrifuged at 13,000RPM for 2 minutes and the supernatant analyzed by HPLC-UV/Vis-radio. De-ethylation of 7-ethoxycoumarin to 7-hydroxycumarin was used as a positive indicator of microsomal P450 activity.
Hepatocytes (HC) in were isolated from male F-344 rats. [14C] TBBA-DBPE (50 and 100M, 190Ci/L) was incubated with HC in suspension (1x106 cells/ mL in 6 well cell culture plates), with shaking (70RPM) at 37oC, or in plated monolayers. Aliquots of media were removed at various times (0-4h: HC suspension; 0-24h: HC monolayers), treated, and analyzed. Conjugation of [14C] TBBA-DBPE and [14C] BPA to [14C] TBBA and [14C] BPA-glucuronide, respectively, was used to confirm metabolic activity.

Details on dosing and sampling:

see section above

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

A single peak that co-eluted with the standard of TBBA-DBPE was detected in extracts of whole blood following oral or IV administration. TBBA-DBPE elimination from the blood was slow. Kinetic constants following IV dosing were: t1/2: 24.8h; CLb: 0.1mL/min. Kinetic constants following oral dosing were: t1/2: 2.5h:13.9h; CLb :4.6mL/min. Systemic bioavailability was 2.2%.

Details on distribution in tissues:

Liver was the major site of disposition following oral or IV administration. After oral administration, 1% of the dose was eliminated in bile in 24h as metabolites. In in vitro experiments utilizing hepatocytes or liver microsomal protein, no detectable metabolism of TBBA-DBPE occurred. These data indicate that TBBA-DBPE is poorly absorbed from the gastrointestinal tract. Compound which is absorbed is sequestered in the liver, slowly metabolized, and eliminated in the feces. (Data od dose recovered from tissues following oral or IV administration of the test substance is presented in Table 2 page 163 of the attached publication).

Details on excretion:

Following a single oral administration of TBBA-DBPE, elimination of [14C] equivalents in feces was extensive and rapid (95% by 96 hours). Following repeated daily oral doses for 5 or 10 days, route and rate of elimination was similar to single administration of TBBA-DBPE. After IV administration, fecal excretion of [14C] equivalents was much slower (27% of dose eliminated by 36h, 71% by 96h). Urinary elimination was minimal (<0.1%) following oral and IV administration.

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#1

Toxicokinetic parameters:

AUC: IV: 52,050 ug/min/mL

Toxicokinetic parameters 2

Test no.:

#2

Toxicokinetic parameters:

AUC: Oral: 951ug/min/mL

Toxicokinetic parameters 3

Toxicokinetic parameters:

other: half life of distribution: 2.5 h

Toxicokinetic parameters 4

Test no.:

#1

Toxicokinetic parameters:

other: IV terminal half life of elimination: 24.8 h

Toxicokinetic parameters 5

Test no.:

#2

Toxicokinetic parameters:

other: Oral terminal half life of elimination: 12.3 h

Toxicokinetic parameters 6

Toxicokinetic parameters:

Cmax: 0.6 ug/mL

Toxicokinetic parameters 7

Toxicokinetic parameters:

Tmax: 7.5 h

Toxicokinetic parameters 8

Toxicokinetic parameters:

other: Systemic clearance from blood(CLb): 0.1mL/min

Toxicokinetic parameters 9

Toxicokinetic parameters:

other: Bioavailability:2.2%

Metabolite characterisation studies

Details on metabolites:

After oral administration, 1% of the dose was eliminated in bile in 24h as metabolites. In in vitro experiments utilizing hepatocytes or liver microsomal protein, no detectable metabolism of TBBA-DBPE occurred. These data indicate that TBBA-DBPE is poorly absorbed from the gastrointestinal tract. Compound which is absorbed is sequestered in the liver, slowly metabolized, and eliminated in the feces.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
The results of these studies show that the likelihood of systemic exposure following ingestion of [14C] TBBA-DBPE is low. Additionally, disposition in liver tissue was minimal, and metabolite formation was slow. Consequently, it was determined that the probability of formation of the carcinogenic moiety DBP was low.
In summary, these data show that TBBA-DBPE was poorly absorbed across the gut lumen following oral administration. However, that which was absorbed was rapidly sequestered in the liver, was slowly metabolized and finally eliminated in the bile for fecal excretion.

Executive summary:

Studies regarding the absorption, distribution, metabolism, and excretion of TBBA-DBPE were conducted. Male Fischer-344 rats were dosed with TBBA-DBPE (20mg/kg) by oral gavage or IV administration. Following a single oral administration of TBBA-DBPE, elimination of [¹⁴C] equivalents in feces was extensive and rapid (95% by 96 hours). Following repeated daily oral doses for 5 or 10 days, route and rate of elimination was similar to single administration of TBBA-DBPE. After IV administration, fecal excretion of [¹⁴C] equivalents was much slower (27% of dose eliminated by 36h, 71% by 96h). Urinary elimination was minimal (<0.1%) following oral and IV administration. A single peak that co-eluted with the standard of TBBA-DBPE was detected in extracts of whole blood following oral or IV administration. TBBA-DBPE elimination from the blood was slow. Kinetic constants following IV dosing were: t_1/2b: 24.8h; CL_b: 0.1mL/min. Kinetic constants following oral dosing were: t_1/2a: 2.5h:13.9h; CL_{b :4.6mL/min.}

Systemic bioavailability was 2.2%. Liver was the major site of disposition following oral or IV administration. After oral administration, 1% of the dose was eliminated in bile in 24h as metabolites. Inin vitroexperiments utilizing hepatocytes or liver microsomal protein, no detectable metabolism of TBBA-DBPE occurred. These data indicate that TBBA-DBPE is poorly absorbed from the gastrointestinal tract. Compound which is absorbed is sequestered in the liver, slowly metabolized, and eliminated in the feces.