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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2012- March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with the principles of GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Objective of study:
absorption
excretion
metabolism
toxicokinetics
other: address Mode of Action of sex-specific liver tumors formed in BDF1 females resulting from long term oral exposure to high biphenyl doses
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Three groups, each consisting of 2 male and female BDF1 mice, were administered 14C-biphenyl via a single oral gavage at 900 mg/kg bw. After oral dosing, excreta (blood, urine, and feces) and selected tissues were collected at different time points. Radioactivity in collected excreta and tissues and the pharmacokinetic parameters for radioactivity in blood were determined. Metabolite profiling and identification were also conducted in urine and blood samples.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): Biphenyl, non radiolabeled 99.9%, biphenyl-14C 98.87%
- Molecular formula (if other than submission substance):not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: suspension
- Physical state: white solid
- Analytical purity: >= 99%
- Impurities (identity and concentrations): <1ppm of As, Cd, Hg, Pb
- Composition of test material, percentage of components: non radiolabeled biphenyl 99.9%
- Isomers composition: no data
- Purity test date: non radiolabeled: 24 Mar 2012
- Lot/batch No.: 120813 (14C) or MKBK0348V (non labelled)
- Expiration date of the lot/batch: no data
- Radiochemical purity (if radiolabelling): Biphenyl-14C with a purity 98.87%
- Specific activity (if radiolabelling): 77 mCi/mmoL
- Locations of the label (if radiolabelling): uniformly labelled in both phenyl rings
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
- Storage condition of test material: non radiolabeled: ambinet, radiolabeled -20C
- Other: Origin of non-radiolabelled Biphenyl: Sigma-Aldrich, Saint Louis. Missouri
- Origin of radiolabelled Biphenyl-C14: American Radiolabeled Company. Saint Louis. Missouri
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
mouse
Strain:
other: BDF1: cross between female C57Bl/6 and male DBA/2
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Supplier and Location: Charles River (Raleigh, North Carolina)
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 25-27 g males, 15-20 g females
- Fasting period before study: no
- Housing: metabolism cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Lab Diet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis. Missouri) in pelleted form
- Water (e.g. ad libitum): municipal water available ad libitum
- Acclimation period: at least 7 days
-Cage type: stainless steel
-Metabolism cages: Roth-type
-Acclimation to metabolism cages: at least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1 °C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/ hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
Administration of a single intraperitoneal dose of 900 mg/kg (vehicle = 0.5% methylcellulose). Observations and sampling up to 7 days after dose administration.
Duration and frequency of treatment / exposure:
single oral dose followed for 7 days or scarified at Cmax of 2 h post-dosing
Doses / concentrations
Remarks:
Doses / Concentrations:
900 mg/kg b.w. containing 1.25 mCi/kg radioactivity
No. of animals per sex per dose:
three groups of 2 animals per sex per dose
Control animals:
no
Positive control:
no
Details on study design:
Three groups of BDF1 male and female mice were given a single oral dose of 900 mg/kg bw biphenyl. Group 1 mice were used for collection of blood for 14C concentration-time course to determine peak (Cmax) blood 14C concentrations. Collection continued for seven days post-dosing. Group 2 mice were sacrificed at Cmax of 2 hrs (determined from Group 1), blood was collected and metabolite analysis performed. Liver was collected and stored. Group 3 mice were used to collect additional excreta over 7 days and selected tissues were harvested for radioactivity and bioconcentration analysis at sacrifice.

Details on dosing and sampling:
-Blood (approximately 5-40uL per time point, Group 1 mice) was collected from the pedal vein at 0.08,0.17,0.25,0.5, 1,2,3,6, 12,24, and every 24 hours post-dosing thereafter from each animal. In order to collect 10-time points, alternate time points/animal were used to determine time-course per sex (5 min, 15 min, 1 hr, 3 hr, and 12 hr) and (10 min, 30 min, 2 hr, 6 hr, 24 hr).
-Blood for Cmax (group 2) was collected at 2 h post-dosing at sacrifice
-Blood and organs from Group 3 animals were collected at sacrifice.
-Blood was oxidized and analyzed by liquid scintillation spectrometry (LSS)
-The urine traps were changed at 12- 24- and 48-hour post-dosing followed by 24-hour intervals for the remainder of the study.
-Faeces were collected at 24-hour intervals.
-Charcoal traps were changed at 24-hour intervals to trap expired volatiles.
-Metabolites were identified with LC/Q-TOF/MS-MS or other LC/MS-MS techniques.
Statistics:
Descriptive statistics were used, i.e., mean ± standard deviation.

Results and discussion

Preliminary studies:
no preliminary studies
Main ADME resultsopen allclose all
Type:
absorption
Results:
Biphenyl was rapidly absorbed; highest blood concentration was measured within 2 hours post- dosing. The absorption half life was 0.5 h for males and 0.3 h for females. Overall, at least 81-82% of the administered dose was absorbed from GI.
Type:
metabolism
Results:
Biphenyl was highly metabolized; both sexes showed similar metabolite profiles. In urine, majority of metabolites were glucuronide and sulphate conjugates of hydroxylated biphenyls. Unchanged parent biphenyl was detected at <1% of the dose.
Type:
excretion
Results:
81 and 69% of dose was eliminated in male and female urines, respectively, mostly within 24hrs. 8 and 10% of dose was eliminated in male and female feces, respectively, mostly within 24hrs. Biphenyl was eliminated faster in BDF1 males than in females.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Orally administered biphenyl was rapidly absorbed without any apparent lag time and achieved the highest measured concentration (Cmax) within 2 hours (Tmax) post- dosing. The absorption half life, t½ of 14C-biphenyl-derived radioactivity from male and female blood was 0.5 and 0.3 hours, respectively. Overall, animals showed high absorption of biphenyl (81-82% of the administered dose), based on recovery in urine and rinse, final cage wash, CO2 and non-GI tissues.
Details on distribution in tissues:
Elimination of the radioactivity from blood was biphasic with most of the elimination occurring during the rapid alpha elimination phase (~6 hours). The slower blood elimination t½ beta was 99 and 671 hours in the males and females, respectively. The AUC of radioactivity at the nominal dose of 900 mg/kg bw oral dose was 1793 µg h g-1 (male) and 3102 µg h g-1 (female). The actual dose levels for these Group 1 mice were 988 mg/kg for male and 851 mg/kg for female, affording a dose-corrected AUC for female mice 2-fold higher than males. The slower urinary and blood elimination half-lives, as well as the larger blood AUC value in female mice demonstrate that biphenyl has higher systemic bioavailability in the female mouse vs. male.
After 168 hours post-dosing, all tissues except for blood had quantifiable mean values ranging from 0.315 - 2.884 µg eq./g tissue, including carcass, liver, kidney, skin and GI tract which were equivalent to 0.06 to 0.09% of orally administered dose, indicating no bioaccumulation in the mouse after single dose.
Details on excretion:
Following a single oral dose of 14C-biphenyl, a mean of 81 and 69% of the administered dose was recovered in urine and the rinse of male and female mice, respectively. Radioactivity was rapidly excreted, most occurring within 24 hours post-dosing with 91% of total urinary elimination observed for males and 56% for females, indicating that biphenyl was eliminated much faster in male mice than in female mice.
A mean of 8 and 10% of the administered dose was recovered in the feces of male and female mice, respectively. Within 24 hours post-dosing, 72 and 57% of total fecal elimination of radioactivity occurred for males and females, respectively; this further indicated that biphenyl was eliminated faster in male mice than in female BDF1 mice.
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
Tmax: males: 2 h, females: 0.5 h
Test no.:
#1
Toxicokinetic parameters:
Cmax: males: 102.657 ug/g, females: 114.05 ug/g
Test no.:
#1
Toxicokinetic parameters:
AUC: males: 1793.265 ug*h/g, females: 3101.999 ug*h/g
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: absorption: males: 0.522 h, females: 0.292 h
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: elimination, alpha: males: 5.556 h, females: 6.789 h
Test no.:
#1
Toxicokinetic parameters:
half-life 3rd: elimination, beta: males: 99.108 h, females: 670.949 h

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Urine:
-26 metabolites identified
-conjugation with sulfate and/or glucuronide, or methylation detected
-glucuronide conjugate of mono-hydroxy biphenyl: 38 and 27% of the dose in males and females
-conjugates of mono-hydroxylated bipheny: 45 and 38% of the dose in males and females
-conjugates of di-hyroxylated biphenyl: 30 and 24% of the dose in males and females
-conjugates of tri-hydroxylated biphenyl: 5 and 7% of the dose in males and females
-mono-hydroxy biphenyl: 3- and 4-phenyl phenol at 0.2-1.3% of the dose
-mercapturic acid conjugates of mono- and di-hydroxylated biphenyls: <1% of the dose in males and females
-unchanged biphenyl, 0.2% of the dose

Cmax blood:
-six metabolites identified
-glucuronide and sulfate conjugates of mono-hydroxy biphenyl
-sulfate conjugate of di-hydroxy biphenyl
-unchanged parent biphenyl
-mono-hydroxy biphenyl: 3- and/or 4-phenyl phenol

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Overall, 14C-biphenyl was rapidly absorbed, highly metabolized and quickly eliminated in the BDF1 mouse. Animals showed high absorption of biphenyl of at least 81-82% of the administered dose. The highest measured concentration of radioactivity, Cmax, in the blood occurred at 2 hours post-dosing. Urine was the primary route of elimination of 14C-biphenyl derived radioactivity (69-81% of the administered dose) with the majority eliminated during the first 24 hours post-dosing. Feces accounted for ≤10% of the administered dose. Gender differences in BDF1 mice were observed in the rates of elimination of radioactivity from the blood. The blood area under the curve (AUC) was 1793 µg h g-1 for males and 3102 µg h g-1 for females, indicating that elimination of biphenyl in female BDF1 mice is substantially slower compared to BDF1 males and that females would overall be exposed to higher doses of biphenyl and/or its metabolites compared to males. Biphenyl was shown to be highly metabolized and both sexes showed similar metabolite profiles. In urine samples, approximately 35-50% of the radioactive dose was identified as the glucuronide conjugates of mono-hydroxylated biphenyl, various sulphate and glucuronide conjugates of di-hydroxy biphenyls and tri-hydroxy biphenyls represented half of the urinary activity, sulphate conjugates of mono-hydroxy biphenyl totaled about 5% of the dose, N-acetyl cysteine conjugates of mono-hydroxy biphenyl represented <1% of the dose. Overall, up to 20 other metabolites were identified, each representing <1% to 3% of the dose. After 168 hours post-dosing, levels in the tissues ranged from 0.06 -0.09 % of the orally administered dose from biphenyl test material, indicating no bioaccumulation of biphenyl or metabolites in BDF1 mouse.
Executive summary:

Three groups, each consisting of 2 male and female BDF1 mice, were administered14C-biphenyl via a single oral gavage at 900 mg/kg bw dose level. After oral dosing, excreta (blood, urine, and feces) and selected tissues were collected at different time points. Radioactivity in collected excreta and tissues was determined and the pharmacokinetic parameters for radioactivity in blood were also derived. Metabolite profiling and identification were also conducted in selected urine and blood sample.

Radioactivity analysis showed that urine was the primary route of elimination of 14C-biphenyl derived radioactivity, through 168 hours post-dosing (69-81% of the administered dose) with the majority eliminated during the first 24 hours post-dosing (56-82% of the total urinary excretion). Feces accounted for ≤10% of the administered dose. 

After 168 hours post-dosing, the potential radioactivity remained in the issues (blood, carcass, liver, kidneys, GI tract, and skin) ranged from 0.06 to 0.09 % of the orally administered dose from biphenyl test material, indicating no bioaccumulation in the mouse.  

     

A blood time-course of radioactivity from males and females administered 900 mg 14C-biphenyl/kg bw showed the test material was rapidly absorbed, with a Tmaxof within 2 hours post-dosing. The elimination of the radioactivity from blood was relatively quick during the alpha elimination phase (t½α~6 hours) followed by a slower elimination during the terminal beta phase (t½b=99 and 671hours). The dose-corrected blood AUC was 2 -fold higher in female mice than male, indicating substantially higher systemic bioavailability of biphenyl and/or metabolites in female mice than in male mice.

The major urinary metabolite, representing approximately 35-50% of the dose, was identified as the glucuronide conjugate of mono-hydroxylated biphenyl.  In addition, there were up to 20 other metabolites tentatively identified, each representing <1% to ~3% of the dose; these included at least 2 isomers of the sulphate conjugate of mono-hydroxy biphenyl, totaling about 5% of the dose.  Total various sulphate and glucuronide conjugates of di-hydroxy biphenyls and tri-hydroxy biphenyls represented half of the urinary activity. The N-acetyl cysteine conjugate of mono-hydroxyl biphenyl in male and female urine represented <1% of the dose.  Unchanged parent biphenyl was detected in the urine samples, representing <1% of the dose.  Little evidence of non-conjugated hydroxylated biphenyl in the urines was found. 

Overall,14C-biphenyl was found to be rapidly absorbed, highly metabolized and quickly eliminated in the BDF1 mouse. Although absorption was comparable between genders, systemic exposure to biphenyl and/or metabolites was thus substantially higher in the female mouse, and is consistent with the observed increased incidence of liver tumors in BDF1 females at high biphenyl doses in the Umeda et al., 2005 cancer bioassay. The prolonged exposure to reactive biphenyl metabolites would result in sex-specific liver injury followed by tissue regeneration and thus be consistent with the threshold MOA for liver tumors reported for the BDF1 female mice.