4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol; bisphenol AF

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study cannot be subsumed under a testing guideline, but is nevertheless well documented and scientifically acceptable.

Objective of study:

bioaccessibility (or bioavailability)

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
The current studies were undertaken to generate TK data for free BPAF (unconjugated parent) and total BPAF (unconjugated and con- jugated BPAF) following oral and IV administration in Harlan Sprague Dawley (HSD) rats and B6C3F1/N mice, the two rodent strains used in the NTP toxicology studies.

- Short description of test conditions:
Toxicokinetics and bioavailability study of bisphenol AF (BPAF) in male / female Harlan Sprague Dawley rats and B6C3F1/N mice following a single gavage. To aid in the interpretation of oral data and to gen- erate oral bioavailability, limited studies were also conducted following an intravenous (IV) dose of 34 mg/kg in male and female rats and mice. Target times for blood collection following the dose administration were 0 (predose), 5, 15, 30 min, 1,2,4,8,12,24,32, 48 h for all dose groups.

- Parameters analysed / observed:
Evaluation focussed on plasma only. A validated analytical method was used to quantitate free (unconjugated parent) and total (unconjugated and conjugated) BPAF in plasma.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: lot # 20100425, 3B Pharmchem International (Wuhan) Co., Ltd. (China).
- Purity, including information on contaminants, isomers, etc.: The purity was determined to be > 99 % (based on high-performance liquid chromatography (HPLC) with ultraviolet detection (UV) at 210nm, and differential scanning calorimetry). < 0.1 % of water is was noted.

Radiolabelling:

no

Remarks:

A method employing protein precipitation followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to quantitate free and total BPAF in plasma. A full validation was conducted in female HSD rat plasma for BPAF.

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Selection of Harlan Sprague Dawley (HSD) rats and B6C3F1/N mice was made as they are the two rodent strains used in the NTP toxicology studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: B6C3F1/N mice were obtained from Taconic Farms (Germantown, NY)
- Age at study initiation: 10-11 weeks(mice)
- Weight at study initiation: mice: Males (25.5-30.5g) Female (18.9-25.2g)
- Housing: n/a - Animals were housed in facilities that are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal procedures were in ac- cordance with the “Guide for the Care and Use of Laboratory Animals” (National Research Council, 2011).
- Diet (e.g. ad libitum): NTP 2000 feed (Ziegler Bros, Inc., Gardners, PA)
- Water (e.g. ad libitum): tap water (Durham, NC)
- Acclimation period: at least 7 days.
- Health status: n/a

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 72 ± 3 °F (22 ± 2 °C)
- Humidity (%): 50 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle.
- Fasting period: not reported

- Other test system relevant information: Enviro Dri packs 100% virgin kraft paper shreds (Sheperd Specialty Papers, Watertown, TN) were provided for environmental enrichment.

IN-LIFE DATES: From: To:Not reported.

Route of administration:

other: 1) ORAL (Gavage); 2) IV

Vehicle:

corn oil

Details on exposure:

ORAL EXPOSURE:
Single oral doses were administered at 34, 110, or 340 mg/kg. Dose formulations were administered in a volume of 5 mL/kg for rat and 10 mL/kg for mouse by intragastric gavage using a syringe equipped with a ball-tipped gavage needle (16G for rats, 18G for mice).

IV EXPOSURE:
Single IV dose was administered at 34 mg/kg; dose was administered in a volume of 2 mL/kg for rats and 4 mL/kg for mice into a lateral tail vein using a syringe equipped with a 27G needle for rats and 30G needle for mice.

FORMULATION ANALYSIS:
Oral dose formulations of BPAF (3.4 mg/mL for mice and 6.8, 22, and 68 mg/mL for rats) were prepared in corn oil and analyzed using a validated HPLC-UV method (linear range, 0.3 to 136 mg/mL; r ≥ 0.99; precision ≤5%; accuracy, ≤ ± 10%). IV dose formulations of BPAF (8.5 mg/mL for mice and 17 mg/mL for rats) were prepared in water:Cremophor:ethanol (67:23:10) and analyzed using a validated HPLC-UV method (linear range, 1 to 20mg/mL r≥0.99; precision ≤5%; accuracy, ≤ ± 10%). All oral and IV formulations were within 10% of the target concentration. Prior to study initiation, stability (≤10% of day 0) of both oral and IV formulations was confirmed for up to 42 d when stored in sealed clear bottles with Teflon-lined lids at ambient or refrigerated conditions.

Duration and frequency of treatment / exposure:

48 hours in total.
Timepoints: 1, 2,4,10,20,24,30,40,48 hours.

Dose / conc.:

34 mg/kg bw/day

Remarks:

ORAL: Single Dose only

Dose / conc.:

34 mg/kg bw/day

Remarks:

IV: Single Dose only

No. of animals per sex per dose / concentration:

3 per sex per dose

Control animals:

no

Positive control reference chemical:

n/a

Details on study design:

- Dose selection rationale: not stated.

- Rationale for animal assignment (if not random):n/a

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: from 5 to 30 minutes; from 1, 24, to 48 hours for all dose groups.

ANALYTICAL METHOD
- Complete description including: limit of detection and quantification, variability and recovery efficiency, matrix used for standard preparations, internal standard. 10% nominal.

A method employing protein precipitation followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to quantitate free and total BPAF in plasma. A full validation was conducted in female HSD rat plasma for BPAF. The validation included an assessment of linearity, inter- and intra-day precision (esti- mated as relative standard deviation, RSD), inter- and intra-day accuracy (estimated as relative standard error, RE), absolute recovery, and experimental limits of quantitation (LOQ) and detection (LOD). Dilution verification was conducted to demonstrate that concentrations outside the validated range could be accurately quantitated after dilution with blank plasma into the validated range. The method was as- sessed for total BPAF (BPAF recovery following deconjugation) by preparing quality control (QC) samples in female HSD rat plasma (n = 6) under the enzyme deconjugation procedure (see below). The method was also assessed for male rat and male and female B6C3F1/N mouse plasma by preparing QC samples in respective matrix (n = 6 for each matrix).

Two stock solutions of BPAF were prepared in acetonitrile and further diluted in the same solvent to generate concentrations of standards in the working range. Stock solutions of DFBPA ((< 0.1%). 2,2-bis(3,5-Difluoro-4-hydroxyphenyl)-hexa- fluoropropane (DFBPA, lot # 20110525, purity 97%) to be used as internal standard (IS)) to be used as IS was prepared in acetonitrile and diluted in acetonitrile to generate working IS solutions. An eight-point solvent calibration curve (~3 to 100 ng/mL) in acetonitrile was prepared using alternate stock solutions. Eight-point matrix calibration curves (~3 to 100 ng/mL) were prepared in duplicate by adding BPAF in female HSD rat plasma, using alternate stock solutions. QC samples were prepared at three concentrations in female HSD rat plasma at 3 levels (~8, 30, and 90 ng/mL, n = 6 at each level per analysis day) using a procedure similar to that for the matrix standards, using an independent stock solution. Matrix blanks were prepared the same as matrix standards except the addition of the analyte.

For the determination of free BPAF, 50 μL aliquots of plasma (matrix calibration standards, QC samples, or matrix blanks) were transferred to microcentrifuge tubes. For the determination of total BPAF, the samples were prepared as follows: to 50 μL aliquots of plasma in mi- crocentrifuge tubes, 10 μL of 0.9% sodium chloride and 25 μL of 190 mM sodium acetate buffer (pH 5) were added along with 10 μL of β-glucuronidase from Helix pomatia and samples were incubated at ~37 °C overnight. To all samples, acetonitrile (150 μL for free and 250 μL for total assessment) was added, and samples were vortexed for 30 s and centrifuged for 5 min. To 125-μL aliquot of the supernatant, 10 μL of 750 ng/mL IS solution was added and analyzed by LC-MS/MS as described below.

Stability of BPAF in extracted samples from above was evaluated when stored at ambient and refrigerated temperatures. Stability of the BPAF in plasma was evaluated following three freeze-thaw cycles and when stored at −70 °C for up to 9 months to cover the study sample storage conditions and duration.

All standards and samples were analyzed by LC-MS/MS using a Shimadzu (Columbus, MD) liquid chromatograph coupled to an Applied Biosystems API-4000 Q-Trap (Waltham, MA) mass spectrometer. Chromatography was performed using a Phenomenex Luna column (C18, 5μm, 50×2.0mm; Santa Clara, CA). Mobile phases A (0.5% formic acid) and B (methanol with 0.5% formic acid) were run with a linear gradient from 30% B to 100% B over 4 min followed by a 4 min hold at a flow rate of 0.3mL/min. The column temperature was maintained at 40 °C. The turbospray ion source was operated in nega- tive mode with a source temperature of 400 °C and an ion spray voltage of −4500 V. Transitions monitored were 335.0 - > 264.7 for BPAF, and 407.0 - > 337.0 for DFBPA. The retention times were 6.3 min for BPAF and 6.7 min for DFBPA.

A linear regression with 1/X weighing was used to relate LC-MS/MS peak area response ratio of analyte to IS and concentration of BPAF in plasma. The concentration of free and total BPAF was calculated using response ratio, the regression equation, initial sample volume, and dilution when applicable.

Statistics:

Individual animal data were evaluated for aberrant concentrations and time points. The actual blood collection times were within 10% from nominal time and hence the actual collection time was used for TK analysis. All concentrations were evaluated to identify outliers by per- forming Q-tests. Based on these assessments, only one value was eliminated from TK analysis.
WinNonlin (Version 6.4, Certara, Princeton, NJ) was used for TK analysis. A variety of compartmental models were tested for BPAF concentration versus time data sets. For each compartmental model, data sets were analyzed with and without weighting. The model and the weighing factor that resulted in the best goodness of fit (evaluated using the Akaike Information Criterion (AIC) and Schwarz Bayesian Criterion (SBC)) was selected as the final model. Based on this, a two-compart- ment model with first order input, first order output and 1/y (1/y2 weighting used for total BPAF 340 mg/kg group) weighting was used to calculate TK parameters following gavage administration (Model 11, Eq. (1)) and a two-compartment model with bolus input, first order output and 1/y2 weighting was used to calculate TK parameters fol- lowing IV administration.

C(t) =Ae t +Be t +Ce k01t (1) C(t) = Ae t + Be t

Based on this, a two-compartment model with first order input, first order output and 1/y (1/y2 weighting used for total BPAF 340 mg/kg group) weighting was used to calculate TK parameters following gavage administration and a two-compartment model with bolus input, first order output and 1/y2 weighting was used to calculate TK parameters following IV administration.
C(t) =Ae t +Be t +Ce -k01t
C(t) = Ae t + Be t

The software used is WinNonlin (Version 6.4, Certara, Princeton, NJ).

Type:

absorption

Results:

Please see 'any other information on results' section for summary

Details on absorption:

Following Oral exposure, BPAF was absorbed rapidly following gavage administration in male and fe- male mice with free BPAF Cmax reached at 0.455h and 0.342h, respectively. Cmax for males and females were 64.1 and 105 ng/mL, respectively.
Following IV administration of 34 mg/kg BPAF in male and female mice, free BPAF levels in plasma were above the LOD in all samples from 5 min through 12 h (female) or 24 h (male) and in some samples at 32 h; levels were below the LOD at 48 h post administration in both males and females.

Details on distribution in tissues:

The volume of distribution was high and exceeded the reported a body water volume in mice (725 mL/kg) indicating distribution of free BPAF into the peripheral compartment. BPAF was cleared rapidly from plasma with an elimination half-life (K10 half-life) of 4.22 and 1.33 h for males and females, re- spectively. AUC0-∞ was similar in males and females.

Details on excretion:

Not measured

Metabolites identified:

not measured

Details on metabolites:

Although metabolites were identified previously, they were not investigated in this study.

Enzymatic activity measured:

Not measured

Bioaccessibility (or Bioavailability) testing results:

Absolute bioavailability of BPAF following gavage administration was estimated in male and female rats and mice using AUC0-∞ of free BPAF following gavage, adjusted for dose administered. In general, absolute bioavailability was very low in both species and sexes. In rats, absolute bioavailability was ~1% with no dose-related effect. In mice, following administration of 34 mg/ kg, absolute bioavailability was slightly higher with ~6 and 3%, in males and females, respectively.
Absolute bioavailability of BPAF following gavage administration was estimated in male and female rats and mice using AUC0-∞ of free BPAF following gavage and IV administration, adjusted for dose ad- ministered. In general, absolute bioavailability was very low in both species and sexes. In rats, absolute bioavailability was ~1% with no dose-related effect. In mice, following administration of 34 mg/ kg, absolute bioavailability was slightly higher with ~6 and 3%, in males and females, respectively.

Analytical method validation

An analytical method to quantitate free BPAF in female HSD rat plasma was developed and successfully validated. A summary of validation parameters investigated and corresponding results are shown in Table 2. The method was linear (≥0.99), accurate (inter-day and intra- day %RE ≤ ± 13.6) and precise (inter-day and intra-day %RSD ≤ 7.1). Experimental LOQ was 2.8 ng/mL and LOD was 0.9 ng/mL. Standards as high as 100,000 ng/mL in plasma could successfully be diluted into the validated concentration range with observed %RE ≤ ± 15.3 and % RSD ≤ 2.7%. The method was acceptable to quantitate total BPAF in plasma with %RE ≤ ± 15.4 and %RSD ≤ 4.1%. The method was qualified to quantitate free BPAF in male HSD rat plasma (%RE, ≤ ± 10.4: %RSD, 5.8%) and male (%RE, ≤ ± 11.6: %RSD, 3.6) and female (%RE, ≤ ± 17.7: %RSD, 7.5) B6C3F1/N mouse plasma using spiked QC standards prepared at 25ng/mL and analyzed with an 8-point standard curve prepared in female HSD rat plasma over the range of ~3 to 100 ng/mL (Table 2).

 

Stability of analytes in extracted samples were demonstrated when stored at ambient temperature or refrigerator at 3 and 90 ng/mL (% RE ≤ ± 18.5). Analyte stability in plasma was confirmed during 3 freeze-thaw cycles at 3, 30, and 90 ng/mL (%RE ≤ ± 11.5) or when stored ~ −70 °C for at least 9 months at 5 and 70 ng/mL (≥95.5% of target) (Table 2). These data confirm that the analytical method was suitable to quantitate free and total BPAF in rat and mouse plasma.

 

Free and total BPAF toxicokinetics in mouse

Following gavage administration, free BPAF was below the LOD for a few animals at later timepoints. Total BPAF was detected above LOD at all timepoints for female mice but was above the LOD only through 32 h for male mice. Plasma concentration versus time data were fitted using a two compartment model with first order input and first order output and 1/y weighting.

 

BPAF was absorbed rapidly following gavage administration in male and female mice with free BPAF Cmax reached at 0.455h and 0.342h, re- spectively. Cmax for males and females were 64.1 and 105 ng/mL, re- spectively. The volume of distribution was high and exceeded the reported a body water volume in mice (725 mL/kg) (Davies and Morris, 1993) indicating distribution of free BPAF into the peripheral com- partment. BPAF was cleared rapidly from plasma with an elimination half-life (K10 half-life) of 4.22 and 1.33 h for males and females, re- spectively. AUC0-∞ was similar in males and females.

 

Total BPAF Cmax and AUC were ≥ 30-fold and ≥ 12-fold higher, respectively, than free BPAF following gavage administration of 34 mg/ kg BPAF in mice. In addition, Cmax was reached ≤0.298 h. These data demonstrate rapid and extensive conjugation of BPAF fol- lowing gavage administration in mice. Total BPAF was cleared from plasma with an elimination half-life (K10 half-life) of 0.753 and 0.804 h for male and females, respectively.

 

Following IV administration of 34 mg/kg BPAF in male and female mice, free BPAF levels in plasma were above the LOD in all samples from 5 min through 12 h (female) or 24 h (male) and in some samples at 32 h; levels were below the LOD at 48 h post administration in both males and females. Total BPAF levels were above the LOD at all time- points. Plasma concentration versus time data were fitted using a two- compartment model with first order input and first order output with 1/ y2 weighting and corresponding TK parameters for free and total BPAF. Free and total BPAF Cmax were similar between male and female mice. However, free BPAF AUC0-∞ was 2-fold and total BPAF AUC0-∞ was 3-fold higher in female mice than male mice. In male mice, plasma elimination half-lives (K10 half-life) of free (0.698 h) and total (1.31 h) BPAF were higher than the corresponding free (0.119 h) and total (0.339 h) values in females.

 

Table 7.1.1/3: Plasma toxicokinetic parameters of free BPAF following a single gavage administration in male and female B6C3F1/N mice^a

Mice B6C3F1/N	Free BPAF	Total BPAF
Sex: Male
Cmax(ng/mL)	64.1 (18.9)	1930 (449)
Tmax(h)	0.455 (0.214)	0.298 (0.071)
K01 Half-life(h)	0.0807 (0.0747)	0.186 (42.4)
K12(1/h)	0.0271 (0.476)	2.42 (635)
K21(1/h)	0.369 (5.1)	0.487 (0.542)
Alpha Half-life(h)	1.67 (21.9)	0.187 (42.7)
Beta Half-life(h)	4.73 (5.8)	5.73 (3.16)
K10 Half-life(h)	4.22 (2.34)	0.753 (171)
Cl 1_F(L/h/kg)	80.0 (22.5)	6.41 (1.94)
Cl2_F(L/h/kg)	13.2 (226)	16.8 (586)
V1_F(L/kg)	487 (238)	6.97 (1590)
V2_F(L/kg)	35.8 (285)	34.6 (1170)
AUC0-∞(h*ng/mL)	425 (119)	5300 (1600)
Sex: Female
Cmax(ng/mL)	105 (20.3)	3970 (1060)
Tmax(h)	0.342 (0.0703)	0.275 (0.101)
K01 Half-life(h)	0.208 (66.9)	0.170 (13.9)
K12(1/h)	2.38 (899)	2.74 (260)
K21(1/h)	0.487 (0.497)	0.536 (0.557)
Alpha Half-life(h)	0.209 (67.2)	0.172 (14.2)
Beta Half-life(h)	9.02 (4.74)	6.03 (3.27)
K10 Half-life(h)	1.33 (426)	0.804 (65.8)
Cl 1_F(L/h/kg)	68.7 (22.5)	2.95 (1.08)
Cl2_F(L/h/kg)	313 (17400)	9.38 (122)
V1_F(L/kg)	131 (42200)	3.43 (281)
V2_F(L/kg)	642 (35300)	17.5 (216)
AUC0-∞(h*ng/mL)	495 (162)	11,500 (4210)

^a Free: Based on two-compartment model with first order input, first order output and 1/y weighting. Total: Based on2two-compartment model with first order input, first order output and 1/y (1/y for 340 mg/kg groups) weighting.

^b Values given are mean (standard error)

 

Table 7.1.1/4:  Plasma toxicokinetic parameters for free and total BPAF following a single intravenous administration of 34 mg/kg BPAF in male and female B6C3F1/N mice^a.

 

Parameter^b	Free		Total
	Male	Female	Male	Female
Cmax (ng/mL)	7490 (1750)	92,300 (32300)	11,700 (2540)	140,000 (29800)
K12 (1/h)	0.189 (0.0802)	0.696 (0.190)	0.126 (0.0997)	0.572 (0.148)
K21 (1/h)	0.227 (0.0304)	0.257 (0.0225)	0.213 (0.0622)	0.263 (0.0277)
Alpha Half-life (h)	0.566 (0.121)	0.106 (0.0193)	0.976 (0.308)	0.259 (0.0366)
Beta Half-life (h)	3.76 (0.316)	3.03 (0.218)	4.37 (0.594)	3.45 (0.209)
K10 Half-Life (h)	0.698 (0.135)	0.119 (0.0225)	1.31 (0.269)	0.339 (0.0478)
Cl1 (L/h/kg)	4.51 (0.547)	2.15 (0.426)	1.54 (0.223)	0.496 (0.058)
Cl2 (L/h/kg)	0.860 (0.324)	0.256 (0.108)	0.367 (0.267)	0.139 (0.0422)
V1 (L/kg)	4.54 (1.06)	0.368 (0.129)	2.91 (0.634)	0.242 (0.0517)
V2 (L/kg)	3.78 (1.10)	0.995 (0.388)	1.73 (0.841)	0.528 (0.125)
Vss	8.32 (1.64)	1.36 (0.499)	4.64 0.994	0.770 0.163
AUC0-∞ (h*ng/mL)	7540 (912)	15,800 (3130)	22,100 (3190)	68,500 (8000)

 

a Based on two-compartment model with first order input, first order output and 1/y weighting.

b Values given are mean (standard error).

 

Table 7.1.1/5: Bioavailability of free BPAF following a single gavage administration in Harlan Sprague Dawley rats and B6C3F1/N mice.

Dose (mg/kg)	Bioavailability (%)^a
	Male rats	Female rats	Male mice	Female mice
34	0.88	1.02	5.64	3.13
110	0.84	1.00
340	1.07	1.06

^a Bioavailability (%F) was calculated as AUC/Dose (oral) ÷ AUC/Dose (IV) x 100.

Table: Values given are mean (standard error)

 

Conclusions:

BPAF is rapidly absorbed in male and female rats and mice following gavage administration and extensively conjugated leading to very low bioavailability (≤6%). BPAF was ra- pidly eliminated in rats and mice with half-lives ≤4.22 h. There were minimal dose, species- or sex-related effects on the plasma toxicokinetics of free BPAF in rats and mice.

Executive summary:

Introduction

This paper reported investigation of the toxicokinetics and bioavailability of bisphenol AF (BPAF) in male and female Harlan Sprague Dawley rats and B6C3F1/N mice following a single gavage administration of 34, 110, or 340 mg/kg, and single IV administration of 34 mg/kg bw. A validated analytical method was used to quantitate free (unconjugated parent) and total (unconjugated and conjugated) BPAF in plasma.

 

Results

Following Oral administration, BPAF was rapidly absorbed in rats with the maximum plasma concentration, Cmax, of free BPAF reached at ≤2.20 h. BPAF was cleared rapidly with a plasma elimination half-life of ≤3.35 h. Cmax and the area under the concentration versus time curve, AUC0-∞, increased proportionally to the dose. Total BPAF Cmax was reached ≤1.07 h in rats with both Cmax (≥27-fold) and AUC0-∞ (≥52-fold) much higher than corresponding free values demonstrating rapid and extensive conjugation of BPAF following oral administration. Absorption of BPAF following a 34 mg/kg gavage dose in mice was more rapid than in rats with free BPAF Cmax reached ≤0.455 h. Free BPAF was cleared rapidly in mice with an elimination half-life of ≤4.22 h. Similar to rats, total BPAF was much higher than corresponding free BPAF. There was no apparent sex-related effect in plasma toxicokinetic parameters of free or total BPAF in mice and rats. 

 

In rats, free and total BPAF was detected at all timepoints in both male and female plasma following IV administration of 34 mg/kg BPAF. Both free and total Cmax and AUC0-∞ were similar between sexes. In male rats, plasma elimination half-lives (K10 half-life) of free and total BPAF were 0.412 h and 0.703 h and were similar between sexes.

 

In male and female mice, free BPAF levels in plasma were above the LOD in all samples from 5 min through 12 h (female) or 24 h (male) and in some samples at 32 h; levels were below the LOD at 48 h post administration in both males and females. Total BPAF levels were above the LOD at all timepoints. Free and total BPAF Cmax were similar between male and female mice. However, free BPAF AUC0-∞ was 2-fold and total BPAF AUC0-∞ was 3-fold higher in female mice than male mice. In male mice, plasma elimination half-lives (K10 half-life) of free (0.698 h) and total (1.31 h) BPAF were higher than the corresponding free (0.119 h) and total (0.339 h) values in females.

 

Absolute bioavailability of BPAF following gavage administration was estimated in male and female rats and mice using AUC0-∞ of free BPAF following gavage and IV administration, adjusted for dose administered. In general, absolute bioavailability was very low in both species and sexes (Table 8). In rats, absolute bioavailability was ~1% with no dose-related effect. In mice, following administration of 34 mg/ kg, absolute bioavailability was slightly higher with ~6 and 3%, in males and females, respectively.

 

Conclusion

These data demonstrate that BPAF was rapidly absorbed following gavage administration in rodents, rapidly and extensively conjugated with low bioavailability.

 

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study cannot be subsumed under a testing guideline, but is nevertheless well documented and scientifically acceptable.

Objective of study:

bioaccessibility (or bioavailability)

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
The current studies were undertaken to generate TK data for free BPAF (unconjugated parent) and total BPAF (unconjugated and con- jugated BPAF) following oral and IV administration in Harlan Sprague Dawley (HSD) rats and B6C3F1/N mice, the two rodent strains used in the NTP toxicology studies.

- Short description of test conditions:
Toxicokinetics and bioavailability study of bisphenol AF (BPAF) in male / female Harlan Sprague Dawley rats and B6C3F1/N mice following a single gavage. To aid in the interpretation of oral data and to gen- erate oral bioavailability, limited studies were also conducted following an intravenous (IV) dose of 34 mg/kg in male and female rats and mice. Target times for blood collection following the dose administration were 0 (predose), 5, 15, 30 min, 1,2,4,8,12,24,32, 48 h for all dose groups.

- Parameters analysed / observed:
Evaluation focussed on plasma only. A validated analytical method was used to quantitate free (unconjugated parent) and total (unconjugated and conjugated) BPAF in plasma.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: lot # 20100425, 3B Pharmchem International (Wuhan) Co., Ltd. (China).
- Purity, including information on contaminants, isomers, etc.: The purity was determined to be > 99 % (based on high-performance liquid chromatography (HPLC) with ultraviolet detection (UV) at 210nm, and differential scanning calorimetry). < 0.1 % of water is was noted.

Radiolabelling:

no

Remarks:

A method employing protein precipitation followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to quantitate free and total BPAF in plasma. A full validation was conducted in female HSD rat plasma for BPAF.

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Selection of Harlan Sprague Dawley (HSD) rats and B6C3F1/N mice was made as they are the two rodent strains used in the NTP toxicology studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rats from Harlan Laboratories (Indianapolis, IN);
- Age at study initiation: 9-10 weeks (rats)
- Weight at study initiation: [Rats: Males (280- 315g) Female (190-237g)]
- Housing: n/a - Animals were housed in facilities that are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal procedures were in accordance with the “Guide for the Care and Use of Laboratory Animals” (National Research Council, 2011).
- Diet (e.g. ad libitum): Rats with 5 K96 (Purina Mills)
- Water (e.g. ad libitum): tap water (Durham, NC)
- Acclimation period: at least 7 days.
- Health status: n/a

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 72 ± 3 °F (22 ± 2 °C)
- Humidity (%): 50%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle.
- Fasting period: not reported

- Other test system relevant information: Enviro Dri packs 100% virgin kraft paper shreds (Sheperd Specialty Papers, Watertown, TN) were provided for environmental enrichment.

IN-LIFE DATES: From: To: Not reported.

Route of administration:

other: 1) Oral: gavage and 2) IV

Vehicle:

corn oil

Details on exposure:

ORAL EXPOSURE:
Single oral doses were administered at 34, 110, or 340 mg/kg. Dose formulations were administered in a volume of 5 mL/kg for rat and 10 mL/kg for mouse by intragastric gavage using a syringe equipped with a ball-tipped gavage needle (16G for rats, 18G for mice).

IV EXPOSURE:
Single IV dose was administered at 34 mg/kg; dose was administered in a volume of 2 mL/kg for rats and 4 mL/kg for mice into a lateral tail vein using a syringe equipped with a 27G needle for rats and 30G needle for mice.

FORMULATION ANALYSIS:
Oral dose formulations of BPAF (3.4 mg/mL for mice and 6.8, 22, and 68 mg/mL for rats) were prepared in corn oil and analyzed using a validated HPLC-UV method (linear range, 0.3 to 136 mg/mL; r ≥ 0.99; precision ≤5%; accuracy, ≤ ± 10%). IV dose formulations of BPAF (8.5 mg/mL for mice and 17 mg/mL for rats) were prepared in water:Cremophor:ethanol (67:23:10) and analyzed using a validated HPLC-UV method (linear range, 1 to 20mg/mL r≥0.99; precision ≤5%; accuracy, ≤ ± 10%). All oral and IV formulations were within 10% of the target concentration. Prior to study initiation, stability (≤10% of day 0) of both oral and IV formulations was confirmed for up to 42 d when stored in sealed clear bottles with Teflon-lined lids at ambient or refrigerated conditions.

Duration and frequency of treatment / exposure:

48 hours in total.
Timepoints: 1, 2,4,10,20,24,30,40,48 hours.

Dose / conc.:

34 mg/kg bw/day (actual dose received)

Remarks:

ORAL: Single Dose only

Dose / conc.:

110 mg/kg bw/day (actual dose received)

Remarks:

ORAL: Single Dose only

Dose / conc.:

340 mg/kg bw/day (actual dose received)

Remarks:

ORAL: Single Dose only

Dose / conc.:

34 mg/kg bw/day (actual dose received)

Remarks:

IV: Single Dose only

No. of animals per sex per dose / concentration:

3 per sex per dose

Control animals:

no

Positive control reference chemical:

n/a

Details on study design:

- Dose selection rationale: not stated.

- Rationale for animal assignment (if not random):n/a

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: from 5 to 30 minutes; from 1, 24, to 48 hours for all dose groups.

ANALYTICAL METHOD
- Complete description including: limit of detection and quantification, variability and recovery efficiency, matrix used for standard preparations, internal standard. 10% nominal.

A method employing protein precipitation followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to quantitate free and total BPAF in plasma. A full validation was conducted in female HSD rat plasma for BPAF. The validation included an assessment of linearity, inter- and intra-day precision (esti- mated as relative standard deviation, RSD), inter- and intra-day accuracy (estimated as relative standard error, RE), absolute recovery, and experimental limits of quantitation (LOQ) and detection (LOD). Dilution verification was conducted to demonstrate that concentrations outside the validated range could be accurately quantitated after dilution with blank plasma into the validated range. The method was as- sessed for total BPAF (BPAF recovery following deconjugation) by preparing quality control (QC) samples in female HSD rat plasma (n = 6) under the enzyme deconjugation procedure (see below). The method was also assessed for male rat and male and female B6C3F1/N mouse plasma by preparing QC samples in respective matrix (n = 6 for each matrix).

Two stock solutions of BPAF were prepared in acetonitrile and further diluted in the same solvent to generate concentrations of standards in the working range. Stock solutions of DFBPA ((< 0.1%). 2,2-bis(3,5-Difluoro-4-hydroxyphenyl)-hexa- fluoropropane (DFBPA, lot # 20110525, purity 97%) to be used as internal standard (IS)) to be used as IS was prepared in acetonitrile and diluted in acetonitrile to generate working IS solutions. An eight-point solvent calibration curve (~3 to 100 ng/mL) in acetonitrile was prepared using alternate stock solutions. Eight-point matrix calibration curves (~3 to 100 ng/mL) were prepared in duplicate by adding BPAF in female HSD rat plasma, using alternate stock solutions. QC samples were prepared at three concentrations in female HSD rat plasma at 3 levels (~8, 30, and 90 ng/mL, n = 6 at each level per analysis day) using a procedure similar to that for the matrix standards, using an independent stock solution. Matrix blanks were prepared the same as matrix standards except the addition of the analyte.

For the determination of free BPAF, 50 μL aliquots of plasma (matrix calibration standards, QC samples, or matrix blanks) were transferred to microcentrifuge tubes. For the determination of total BPAF, the samples were prepared as follows: to 50 μL aliquots of plasma in mi- crocentrifuge tubes, 10 μL of 0.9% sodium chloride and 25 μL of 190 mM sodium acetate buffer (pH 5) were added along with 10 μL of β-glucuronidase from Helix pomatia and samples were incubated at ~37 °C overnight. To all samples, acetonitrile (150 μL for free and 250 μL for total assessment) was added, and samples were vortexed for 30 s and centrifuged for 5 min. To 125-μL aliquot of the supernatant, 10 μL of 750 ng/mL IS solution was added and analyzed by LC-MS/MS as described below.

Stability of BPAF in extracted samples from above was evaluated when stored at ambient and refrigerated temperatures. Stability of the BPAF in plasma was evaluated following three freeze-thaw cycles and when stored at −70 °C for up to 9 months to cover the study sample storage conditions and duration.

All standards and samples were analyzed by LC-MS/MS using a Shimadzu (Columbus, MD) liquid chromatograph coupled to an Applied Biosystems API-4000 Q-Trap (Waltham, MA) mass spectrometer. Chromatography was performed using a Phenomenex Luna column (C18, 5μm, 50×2.0mm; Santa Clara, CA). Mobile phases A (0.5% formic acid) and B (methanol with 0.5% formic acid) were run with a linear gradient from 30% B to 100% B over 4 min followed by a 4 min hold at a flow rate of 0.3mL/min. The column temperature was maintained at 40 °C. The turbospray ion source was operated in nega- tive mode with a source temperature of 400 °C and an ion spray voltage of −4500 V. Transitions monitored were 335.0 - > 264.7 for BPAF, and 407.0 - > 337.0 for DFBPA. The retention times were 6.3 min for BPAF and 6.7 min for DFBPA.

A linear regression with 1/X weighing was used to relate LC-MS/MS peak area response ratio of analyte to IS and concentration of BPAF in plasma. The concentration of free and total BPAF was calculated using response ratio, the regression equation, initial sample volume, and dilution when applicable.

Statistics:

Individual animal data were evaluated for aberrant concentrations and time points. The actual blood collection times were within 10% from nominal time and hence the actual collection time was used for TK analysis. All concentrations were evaluated to identify outliers by per- forming Q-tests. Based on these assessments, only one value was eliminated from TK analysis.
WinNonlin (Version 6.4, Certara, Princeton, NJ) was used for TK analysis. A variety of compartmental models were tested for BPAF concentration versus time data sets. For each compartmental model, data sets were analyzed with and without weighting. The model and the weighing factor that resulted in the best goodness of fit (evaluated using the Akaike Information Criterion (AIC) and Schwarz Bayesian Criterion (SBC)) was selected as the final model. Based on this, a two-compart- ment model with first order input, first order output and 1/y (1/y2 weighting used for total BPAF 340 mg/kg group) weighting was used to calculate TK parameters following gavage administration (Model 11, Eq. (1)) and a two-compartment model with bolus input, first order output and 1/y2 weighting was used to calculate TK parameters fol- lowing IV administration.
C(t) =Ae t +Be t +Ce k01t (1) C(t) = Ae t + Be t

Based on this, a two-compartment model with first order input, first order output and 1/y (1/y2 weighting used for total BPAF 340 mg/kg group) weighting was used to calculate TK parameters following gavage administration and a two-compartment model with bolus input, first order output and 1/y2 weighting was used to calculate TK parameters following IV administration.
C(t) =Ae t +Be t +Ce -k01t
C(t) = Ae t + Be t

The software used is WinNonlin (Version 6.4, Certara, Princeton, NJ).

Preliminary studies:

n/a

Type:

absorption

Results:

Please see 'any other information on results' section for summary

Details on absorption:

BPAF was absorbed rapidly following gavage administration in male and female rats with Tmax reached at ≤2.2 h; there was an apparent increase in Tmax with the increasing dose. In general, Cmax increased proportionally to the dose.

Details on distribution in tissues:

The volume of distribution was high and exceeded the reported body water volume in rats (688mL/kg) indicating distribution of free BPAF into the peripheral compartment. BPAF was cleared rapidly from plasma with an elimination half-life (K10 half-life) ranging from 1.68 to 3.35 h, depending on the dose administered. AUC0-∞ increased proportionally to the dose. There was no apparent sex-related difference in plasma toxicokinetic para- meters of free BPAF in rats.

Details on excretion:

not measured

Metabolites identified:

not measured

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability) testing results:

Absolute bioavailability of BPAF following gavage administration was estimated in male and female rats and mice using AUC0-∞ of free BPAF following gavage, adjusted for dose administered. In general, absolute bioavailability was very low in both species and sexes. In rats, absolute bioavailability was ~1% with no dose-related effect. In mice, following administration of 34 mg/ kg, absolute bioavailability was slightly higher with ~6 and 3%, in males and females, respectively.
Absolute bioavailability of BPAF following gavage administration was estimated in male and female rats and mice using AUC0-∞ of free BPAF following gavage and IV administration, adjusted for dose ad- ministered. In general, absolute bioavailability was very low in both species and sexes. In rats, absolute bioavailability was ~1% with no dose-related effect. In mice, following administration of 34 mg/ kg, absolute bioavailability was slightly higher with ~6 and 3%, in males and females, respectively.

Analytical method validation

An analytical method to quantitate free BPAF in female HSD rat plasma was developed and successfully validated. A summary of validation parameters investigated and corresponding results are shown in Table 2. The method was linear (≥0.99), accurate (inter-day and intra- day %RE ≤ ± 13.6) and precise (inter-day and intra-day %RSD ≤ 7.1). Experimental LOQ was 2.8 ng/mL and LOD was 0.9 ng/mL. Standards as high as 100,000 ng/mL in plasma could successfully be diluted into the validated concentration range with observed %RE ≤ ± 15.3 and % RSD ≤ 2.7%. The method was acceptable to quantitate total BPAF in plasma with %RE ≤ ± 15.4 and %RSD ≤ 4.1%. The method was qualified to quantitate free BPAF in male HSD rat plasma (%RE, ≤ ± 10.4: %RSD, 5.8%) and male (%RE, ≤ ± 11.6: %RSD, 3.6) and female (%RE, ≤ ± 17.7: %RSD, 7.5) B6C3F1/N mouse plasma using spiked QC standards prepared at 25ng/mL and analyzed with an 8-point standard curve prepared in female HSD rat plasma over the range of ~3 to 100 ng/mL (Table 2).

 

Stability of analytes in extracted samples were demonstrated when stored at ambient temperature or refrigerator at 3 and 90 ng/mL (% RE ≤ ± 18.5). Analyte stability in plasma was confirmed during 3 freeze-thaw cycles at 3, 30, and 90 ng/mL (%RE ≤ ± 11.5) or when stored ~ −70 °C for at least 9 months at 5 and 70 ng/mL (≥95.5% of target) (Table 2). These data confirm that the analytical method was suitable to quantitate free and total BPAF in rat and mouse plasma.

 

Free and total BPAF toxicokinetics in rat

Free and total BPAF was detected at all timepoints in both male and female plasma following gavage administration of 34, 110, and 340 mg/kg BPAF. Plasma concentration versus time data were fitted using a two-compartment model with first order input, first order output and 1/y (1/y2 for total BPAF in 340 mg/kg groups) weighting.

 

BPAF was absorbed rapidly following gavage administration in male and female rats with Tmax reached at ≤2.2 h; there was an apparent increase in Tmax with the increasing dose. In general, Cmax increased proportionally to the dose. The volume of distribution was high and exceeded the reported body water volume in rats (688mL/kg) indicating distribution of free BPAF into the peripheral compartment. BPAF was cleared rapidly from plasma with an elimination half-life (K10 half-life) ranging from 1.68 to 3.35 h, depending on the dose administered. AUC0-∞ increased proportionally to the dose. There was no apparent sex-related difference in plasma toxicokinetic parameters of free BPAF in rats.

 

Total BPAF Cmax and AUC were ≥ 27- and ≥ 52-fold higher, respectively, than free BPAF following gavage administration of BPAF in rats. In addition, Cmax was reached ≤1.07 h. These data demon- strate rapid and extensive conjugation of BPAF following gavage ad- ministration in rats. Cmax increased less than proportionally to the dose at the highest dose of 340 mg/kg in both males and females. Total BPAF was cleared from plasma more slowly than free BPAF with an elim- ination half-life (K10 half-life) ranging from 2.60 to 4.61h for all groups except 340 mg/kg female group where it was 20.2 h. AUC0-∞ increased proportionally to the dose in males but not in females where it was more than proportional to the dose.

 

Free and total BPAF was detected at all timepoints in both male and female plasma following IV administration of 34 mg/kg BPAF. Both free and total Cmax and AUC0-∞ were similar between sexes. In male rats, plasma elimination half-lives (K10 half-life) of free and total BPAF were 0.412 h and 0.703 h and were similar between sexes.

 

Table 7.1.1/3: Plasma toxicokinetic parameters of free BPAF following a single gavage administration in male and female Harlan Sprague Dawley rats^a

Parameter^b	Dose(mg/kg) Free BPAF			Dose(mg/kg) Total BPAF
Sex: Male	34	110	340		34	110	340
Cmax(ng/mL)	60.7 (8.39)	142 (31.6)	552 (138)		2750 (525)	7970 (1920)	10,500 (2400)
Tmax(h)	0.812(0.352)	1.43(0.339)	2.20(0.718)		0.734 (0.180)	1.07 (0.386)	3.97 (0.974)
K01 Half-life(h)	0.491 (286)	0.910 (123)	1.44 (381)		0.447 (12)	0.561 (171)	2.43 (129)
K12(1/h)	0.849 (604)	0.402(62.7)	0.156(49.5)		1.13 (35.9)	0.731 (321)	0.0931 (6.73)
K21(1/h)	0.257(0.137)	0.0972(0.207)	0.0814(0.223)		0.276 (0.308)	0.380 (0.568)	0.0682 (0.177)
Alpha Half-life(h)	0.491 (285)	0.918 (125)	1.43 (379)		0.427 (11.2)	0.558 (170)	2.53 (136)
Beta Half-life(h)	10.2 (4.5)	18.4 (35.5)	13.9 (28.1)		15.3 (8.95)	11.9 (6.14)	18.5 (29.7)
K10 Half-life(h)	1.85 (1080)	2.36 (320)	2.33 (619)		2.60 (70.1)	3.65 (1110)	4.61 (244)
Cl 1_F(L/h/kg)	85.2 (20.0)	89.1 (52.4)	70.3 (20.8)		1.32 (0.382)	1.17 (0.314)	1.35 (0.303)
Cl2_F(L/h/kg)	193 (24800)	122 (2520)	36.9 (1910)		5.58 (26.9)	4.51 (603)	0.836 (16.1)
V1_F(L/kg)	227(132000)	304 (41100)	237 (62900)		4.95 (134)	6.18 (1890)	8.98 (476)
V2_F(L/kg)	752(966600)	1260 (23900)	454 (24400)		20.2 (115)	11.9 (1600)	12.3 (208)
AUC0-∞(h*ng/mL)	399 (93.5)	1230 (726)	4830 (1430)		25,700(7450)	93,700 (25100)	252,000(56400)
Sex: Female
Cmax(ng/mL)	47.4 (8.39)	245 (29.3)	555 (99.5)		3760 (695)	10,500 (2400)	15,200 (5530)
Tmax(h)	0.767(0.201)	0.658 (0.1)	1.34 (0.24)		0.410 (0.126)	0.564 (0.166)	2.19 (0.64)
K01 Half-life(h)	0.350 (6.52)	0.405 (6.32)	0.892 (30.4)		0.204 (38.1)	0.352 (17.2)	1.35 (35)
K12(1/h)	0.981 (31.1)	1.07 (19.3)	0.433 (15.4)		2.19 (595)	1.64 (91.5)	0.411 (11.7)
K21(1/h)	0.698 (1.29)	0.215 (0.115)	0.0391(0.0929)		1.06 (0.809)	0.240 (0.216)	0.0498(0.0765)
Alpha Half-life(h)	0.384 (7.67)	0.422 (6.63)	0.912 (31.2)		0.203 (37.9)	0.344 (16.8)	1.41 (36.5)
Beta Half-life(h)	8.68 (2.6)	12.8 (4.83)	44.4 (152)		9.74 (2.08)	38.7 (46.9)	200 (1380)
K10 Half-life(h)	3.35 (61.8)	1.68 (26)	2.29 (79.2)		3.03 (566)	4.59 (225)	20.2 (586)
Cl 1_F(L/h/kg)	73.8 (13.3)	74.7 (13.4)	70.7 (94.0)		0.954 (0.172)	0.623(0.49)	0.309(1.78)
Cl2_F(L/h/kg)	350 (4650)	194 (503)	101 (180)		9.14 (766)	6.78(43.2)	3.70(10.3)
V1_F(L/kg)	357 (6600)	181 (2810)	33 (7920)		4.17 (780)	4.13(204)	9.01 (232)
V2_F(L/kg)	502 (5820)	899 (2050)	2580 (6150)		8.61 (725)	28.3(195)	74.4 (185)
AUC0-∞(h*ng/mL)	461 (82.7)	1470 (264)	4810 (6390)		35,600(6420)	177,000(139000)	1,100,000 (6340000)

^a Free: Based on two-compartment model with first order input, first order output and 1/y weighting. Total: Based on2two-compartment model with first order input, first order output and 1/y (1/y for 340 mg/kg groups) weighting.

^b Values given are mean (standard error)

 

Table 7.1.1/4:  Plasma toxicokinetic parameters for free and total BPAF following a single intravenous administration of 34 mg/kg BPAF in male and female Harlan Sprague Dawley rats^a.

Parameter^b	Free		Total
	Male	Female	Male	Female
Cmax (ng/mL)	75,900 (13700)	71,500 (17300)	96,700 (20000)	130,000 (29000)
K12 (1/h)	0.0503 (0.0151)	0.0656 (0.0398)	0.333 (0.149)	0.340 (0.125)
K21 (1/h)	0.0892 (0.0147)	0.141 (0.0339)	0.128 (0.0235)	0.151 (0.0299)
Alpha Half-life (h)	0.400 (0.039)	0.420 (0.0749)	0.512 (0.113)	0.463 (0.0933)
Beta Half-life (h)	8.01 (1.28)	5.15 (1.15)	7.46 (0.829)	6.13 (0.883)
K10 Half-Life (h)	0.412 (0.0394)	0.439 (0.0724)	0.703 (0.12)	0.620 (0.109)
Cl1 (L/h/kg)	0.753 (0.102)	0.751 (0.118)	0.347 (0.0429)	0.292 0.0322
Cl2 (L/h/kg)	0.0225 (0.00797)	0.0312 (0.0177)	0.117 (0.0498)	0.0887 (0.0294)
V1 (L/kg)	0.448 (0.0811)	0.476 (0.115)	0.352 (0.0729)	0.261 (0.0581)
V2 (L/kg)	0.253 (0.0785)	0.222 (0.0922)	0.917 (0.277)	0.587 (0.143)
Vss	0.700 (0.146)	0.697 (0.166)	1.27 (0.305)	0.848 (0.173)
AUC0-∞ (h*ng/mL)	45,200 (6110)	45,300 (7110)	98,000 (12100)	117,000 (12800)

a Based on two-compartment model with first order input, first order output and 1/y weighting.

b Values given are mean (standard error).

 

Table 7.1.1/5: Bioavailability of free BPAF following a single gavage administration in Harlan Sprague Dawley rats and B6C3F1/N mice.

Dose (mg/kg)	Bioavailability (%)^a
	Male rats	Female rats	Male mice	Female mice
34	0.88	1.02	5.64	3.13
110	0.84	1.00
340	1.07	1.06

^a Bioavailability (%F) was calculated as AUC/Dose (oral) ÷ AUC/Dose (IV) x 100.

Conclusions:

BPAF is rapidly absorbed in male and female rats and mice following gavage administration and extensively conjugated leading to very low bioavailability (≤6%). BPAF was rapidly eliminated in rats and mice with half-lives ≤4.22 h. There were minimal dose, species- or sex-related effects on the plasma toxicokinetics of free BPAF in rats and mice.

Executive summary:

Introduction

This paper reported investigation of the toxicokinetics and bioavailability of bisphenol AF (BPAF) in male and female Harlan Sprague Dawley rats and B6C3F1/N mice following a single gavage administration of 34, 110, or 340 mg/kg, and single IV administration of 34 mg/kg bw. A validated analytical method was used to quantitate free (unconjugated parent) and total (unconjugated and conjugated) BPAF in plasma.

 

Results

Following Oral administration, BPAF was rapidly absorbed in rats with the maximum plasma concentration, Cmax, of free BPAF reached at ≤2.20 h. BPAF was cleared rapidly with a plasma elimination half-life of ≤3.35 h. Cmax and the area under the concentration versus time curve, AUC0-∞, increased proportionally to the dose. Total BPAF Cmax was reached ≤1.07 h in rats with both Cmax (≥27-fold) and AUC0-∞ (≥52-fold) much higher than corresponding free values demonstrating rapid and extensive conjugation of BPAF following oral administration. Absorption of BPAF following a 34 mg/kg gavage dose in mice was more rapid than in rats with free BPAF Cmax reached ≤0.455 h. Free BPAF was cleared rapidly in mice with an elimination half-life of ≤4.22 h. Similar to rats, total BPAF was much higher than corresponding free BPAF. There was no apparent sex-related effect in plasma toxicokinetic parameters of free or total BPAF in mice and rats. 

 

In rats, free and total BPAF was detected at all timepoints in both male and female plasma following IV administration of 34 mg/kg BPAF. Both free and total Cmax and AUC0-∞ were similar between sexes. In male rats, plasma elimination half-lives (K10 half-life) of free and total BPAF were 0.412 h and 0.703 h and were similar between sexes.

 

In male and female mice, free BPAF levels in plasma were above the LOD in all samples from 5 min through 12 h (female) or 24 h (male) and in some samples at 32 h; levels were below the LOD at 48 h post administration in both males and females. Total BPAF levels were above the LOD at all timepoints. Free and total BPAF Cmax were similar between male and female mice. However, free BPAF AUC0-∞ was 2-fold and total BPAF AUC0-∞ was 3-fold higher in female mice than male mice. In male mice, plasma elimination half-lives (K10 half-life) of free (0.698 h) and total (1.31 h) BPAF were higher than the corresponding free (0.119 h) and total (0.339 h) values in females.

 

Absolute bioavailability of BPAF following gavage administration was estimated in male and female rats and mice using AUC0-∞ of free BPAF following gavage and IV administration, adjusted for dose administered. In general, absolute bioavailability was very low in both species and sexes (Table 8). In rats, absolute bioavailability was ~1% with no dose-related effect. In mice, following administration of 34 mg/ kg, absolute bioavailability was slightly higher with ~6 and 3%, in males and females, respectively.

 

Conclusion

These data demonstrate that BPAF was rapidly absorbed following gavage administration in rodents, rapidly and extensively conjugated with low bioavailability.

 

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Only 1 dose rate tested with no justification provided as to why this would be sufficient. Presence of metabolites not mentioned.

GLP compliance:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stock solution (in methanol) stored at -20 ºC.
- Stability under test conditions: Not required (ADME study- with UPLC/MS analysis throughout testing period)
- Solubility and stability of the test substance in the solvent/vehicle: Stock solution prepared at 1 mg/L.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in methanol.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 1 mg/L stock solution prepared, in methanol.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid.

OTHER SPECIFICS: n/a

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Academy of Military Medical Sciences, China.
- Age at study initiation: 8 - 9 weeks old
- Weight at study initiation: 250 - 300 g
- Housing: Housed individually in steel metabolism cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ºC
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12:12 light: dark

IN-LIFE DATES: Not reported

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared from a 1 mg/L methanol stock solution. Details on the preparation of the dose solutions are not reported.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not reported
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

Duration and frequency of treatment / exposure:

Individuals were treated daily for 2 weeks.

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4

Control animals:

yes

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: Approximately 350-fold lower than the published acute LD50 data in rats.
- Rationale for animal assignment (if not random): Not reported

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, kidney, liver, testis, adipose and muscle.
- Time and frequency of sampling: At the end of the treatment period.
- Other: n/a
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, tissues (as above).
- Time and frequency of sampling: 1 sampling interval at the end of the treatment period (Day 14).
- From how many animals: 4
- Method type(s) for identification HPLC-MS-MS
- Limits of detection and quantification: Signal: noise ratio of 3:1 and 10:1 for LOD and LOQ, respectively, yielded quantification levels of 1 µg/kg for liver, muscle, adipose, kidney and urine samples; and 3 µg/kg for faeces.
- Other: n/a
TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): Enzymatic hydrolysis

Statistics:

A statistical analysis was performed by SPSS 16.0 to compare the difference in BPAF concentrations between samples with and without enzymatic hydrolysis, using the Wilcoxon Signed Ranks Test. The results of the analysis would dictate if the enzymatic hydrolysis procedure should be adopted in the test.

Preliminary studies:

Recovery studies were performed at three fortification levels, which were selected according to the sensitivity of each matrix. The mean recoveries ranged from 71.0% to 102.3% with relative standard deviations of no more than 13.2% (n = 6).

Details on absorption:

Bisphenol-AF was poorly adsorbed with the vast majority of the test item excreted in the faeces ( > 10 mg/kg).

Details on distribution in tissues:

Bisphenol-AF was found in highest concentrations in liver and kidney tissues and serum. Maximum concentrations of 1637.5 µg/kg were observed in liver tissue (after enzymatic hydrolysis).

Details on excretion:

Faecal excretion was the major route of elimination, and urinary excretion the secondary route.

Metabolites identified:

no

Details on metabolites:

Metabolites were not formally identified. The majority of bisphenol-AF was present in its conjugate form (> 86 %).

Bioaccessibility (or Bioavailability) testing results:

Not determined

Table 1       Concentration of bisphenol-AF in orally dosed rats

Tissues and excreta	Concentration (µg/kg)
	Without enzymatic hydrolysis				After enzymatic hydrolysis
	Rat 1	Rat 2	Rat 3	Rat 4	Rat 1	Rat 2	Rat 3	Rat 4
Liver	78.1	141.0	437.5	190.5	1210.2	1376.3	1637.5	1496.6
Muscle	8.1	10.0	12.8	17.1	44.3	20.1	17.6	19.5
Adipose	30.3	30.3	39.3	40.1	62.7	47.2	42.1	57.8
Testis	15.3	16.0	21.5	24.0	66.0	50.0	68.0	47.0
Kidney	31.4	28.3	56.3	45.5	880.3	387.0	372.5	431.3
Serum	3.4	3.2	3.9	4.3	1075.3	358.8	312.4	431.3
Urine	31.5	17.0	17.6	25.5	177.6	47.5	25.7	59.8
Faeces	424682.4	449908.6	337844.6	146468.7	545266.9	469864.7	370004.5	223646.9

Conclusions:

Bisphenol-AF was poorly adorbed to the rat GI system where concentrations of test item greater than the application rate were observed in the faeces of individuals, indicating low potential for bioaccumulation. The highest level of bisphenol-AF were found in the liver, kidneys and serum. Extractions performed with and without enzyme hydrolysis indicated that the vast majority of recovered test item was present in its conjugated form, indicative of a test item that is readily metabolised by the liver. Faeces were confirmed as the major route of excretion of the test item with urine a secondary route.

Executive summary:

In a metabolism study bisphenol-AF (98 % purity), was administered to 4 male Sprague-Dawley rats over 14 consecutive days by oral gavage at a dose level of 10 mg/kg bw/day.

High levels of BPAF were detected in the liver, kidney and serum samples. The significant enhancement of the test item concentration after enzymatic hydrolysis in the serum, liver and kidney samples implies that the liver is the major organ responsible for metabolism and that the kidney plays an important part in the excretion of its metabolites. The highest level of bisphenol-AF was observed in the faeces, which indicates that most of the test item was excreted as the nonconjugated form. Faecal excretion was the major route of elimination, and urinary excretion was the secondary route.

Results were determined using a validated method where fortified samples, performed at three fortification levels, were extracted to confirm the sensitivity of the extraction and analytical methods. The mean recoveries ranged from 71.0 % to 102.3 % with relative standard deviations of no more than 13.2 % (n = 6) and the limit of quantification was determied to be 1 µg/kg for liver, muscle, adipose, kidney and urine samples and 3 µg/kg for faeces. Reported values indicate an adequately efficient and sensitive method.

This metabolism study in the rat is classified acceptable and satisfies the guideline requirement for a metabolism study OECD 417 in rats.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Principles of method if other than guideline:

Investigation of the endocrine disrupting potential of bisphenol AF and its metabolite bisphenol AF glucuronide.

GLP compliance:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Test item solubilised in corn oil at a concentration of 100 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in corn oil prior to application
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Stock solution prepared at 100 mg/mL
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid.

OTHER SPECIFICS: n/a

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Academy of Military Medical Sciences, China
- Age at study initiation: 8 weeks
- Weight at study initiation: 250 - 300 g
- Housing: Steel metabolism cages
- Diet (e.g. ad libitum): Rat chow, ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Not reported

IN-LIFE DATES: Not reported

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item disolved in corn oil at 100 mg/mL.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

Duration and frequency of treatment / exposure:

Individuals were dosed daily for 2 weeks

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Repeat dose (daily for 2 weeks) for quantifying metabolite formation in male rats.

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Single dose (for metabolite evaluation)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Single dose (for metabolite evaluation)

No. of animals per sex per dose / concentration:

Metabolite quantification - not reported.
Metabolite evaluation - 3 rats per treatment.

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): Random

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Not conducted

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine
- Time and frequency of sampling: daily
- From how many animals: (samples pooled or not): samples pooled daily
- Method type(s) for identification: UPLC/MS/MS and NMR (BPAF-G only).
- Limits of quantification: 1 µg/L and 10 µg/L for BPAF and BPAF-G, respectively.
- Other: Analytical method extraction efficienty ranged from 82.8 - 106.7 % (RSD: 10.9 %)
TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): Not reported

Statistics:

Statistical analysis was performed using one-way ANOVA followed by a Tukey-Kramer multiple comparison test. A p value < 0.05 was considered statistically significant.

Type:

metabolism

Results:

4 metabolites were observed in rat urine; BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrate and a sulphate conjugation of BPAF. BPAF glucuronide was the most significant metabolite with 100 % of BPAF transforming to BPAF-G.

Details on absorption:

Not studied

Details on distribution in tissues:

Not studied

Details on excretion:

After treatment with a single dose of 20 mg/kg or 100 mg/kg BPAF, the peak concentration of BPAF-G in plasma was observed at 30 min followed by rapid decline in the next three hours, representing quick clearance of BPAF in the rats.

Metabolites identified:

yes

Details on metabolites:

4 metabolites were observed in rat urine; BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrate and a sulphate conjugation of BPAF. BPAF glucuronide was the most significant metabolite with 100 % of BPAF transforming to BPAF-G.

Conclusions:

Four metabolites, including diglucuronide conjugated (M1), glucuronide conjugated (M2), glucuronide dehydrated (M3) and sulfate conjugated (M4), were identified in the urine of SD rats. M1, M2 and M3 were related to glucuronidation indicating that glucuronidation is an important reaction for BPAF metabolism. Among the four metabolites, BPAF-G was the only metabolite detected in the plasma of SD rats administrated with a single dose of BPAF, implying that M1 and M3 may be the byproducts or reactive intermediates produced during BPAF glucuronidation. Sulfate conjugation (M4) is also detected, suggesting that there are other metabolism pathways and different enzymes responsible for BPAF biotransformation in
vivo. However, the value of mass spectrometry response for M4 was substantially lower than that for M2, which suggests that sulfate conjugation is not likely the major pathway for BPAF biotransformation in vivo.

Executive summary:

The biotransformation of bisphenol AF (BPAF) by glucuronidation to BPAF-G was observed in Sprague-Dawley rats. Four metabolites, including diglucuronide conjugated (M1), glucuronide conjugated (M2), glucuronide dehydrated (M3) and sulfate conjugated (M4), were identified in the urine of SD rats. M1, M2 and M3 were related to glucuronidation indicating that glucuronidation is an important reaction for BPAF metabolism. Among the four metabolites, BPAF-G was the only metabolite detected in the plasma of SD rats administrated with a single dose of BPAF, implying that M1 and M3 may be the byproducts or reactive intermediates produced during BPAF glucuronidation. Sulfate conjugation (M4) is also detected, suggesting that there are other metabolism pathways and different enzymes responsible for BPAF biotransformation in vivo. However, the value of mass spectrometry response for M4 was substantially lower than that for M2, which suggests that sulfate conjugation is not likely the major pathway for BPAF biotransformation in vivo.

Description of key information

There was no absorption data so for assessment, 100% absorption for all routes is assumed based on the physico-chemical properties of the test item.

The test item is widely distributed in the tissues mainly kidneys, liver, testis, adipose and muscle.

Metabolism is mainly through liver and intestine - phase I and II enzymes with possible involvement of gut micro flora, glucuronosyltransferases and sulfonyltransferase. Main metabolites includes; BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate.

Elimination via Urine as conjugates and parent compound through faeces.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

Absorption: the molecular weight of BPAF is 336.2 g/mol, it’s n-octanol/water partition coefficient 2.79 at 20 °C, boiling point of >350 °C, solubility of 222.4 mg/L and vapour pressure of 5E-06 Pa are. Although these properties make uptake from all routes possible, for dermal route, the surface tension could restrict transfer between the stratum corneum and the epidermis and therefore overall uptake via this route may be limited. This is demonstrated by the lack of significant systemic toxicity from both sensitisation and local toxicity studies. Oral absorption of the test item is mainly via passive diffusing into portal circulation with delivery into the liver i.e. first pass metabolism. This is demonstrated by the high level of BPAF detected in the liver, kidney and serum of rat exposed to the test item (Yang 2012).

 

Absorption of BPAF following a 34 mg/kg gavage dose in mice was more rapid than in rats with free BPAF Cmax reached ≤0.455 h.Free BPAF was cleared rapidly in mice with an elimination half-life of ≤4.22 h. Similar to rats, total BPAF was much higher than corresponding free BPAF. (Waidyanatha et al. 2019)

 

 

Distribution: the substance has pysiocohemical properitiy that mean the test item can easily dissolve into the gastrointestinal fluid, pass through aqueous pores or be carried through the epithelial barrier by the bulk passage of water into the liver. A wide distribution of the substance via oral route is expected as demonstrated by the content of BPAF in kidneys, liver, testis, adipose and muscle of exposed rats (Yang 2012).

 

Metabolism is mainly through the liver via phase I and II enzymes as demonstrated by the conjugated form of the test item in tissues and urine, for example, a high proportion of BPAF-conjugate was present in liver, kidney and serum samples at approximately 86.0%, 90.9%, and 99.1%, respectively (Yang 2012). Metabolism mainly involves hydrolysis of cyclic alcohol, glucuronidation and sulfation to subsequent conjugates which can also be deconjugated back to parent compound by the intestinal micro flora.  This is supported by the four identified metabolites; BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. The biotransformation of BPAF is rapid as demonstrated by the peak of BPAF-glucuronide in plasma which was observed within 30 minutes followed by rapid decline in the next three hours, representing quick clearance of BPAF in the rats. The peak of BPAF was achieved at 1 h and eliminated completely at 48 h. BPAF glucuronide considered as the major metabolite in vivo (Li 2013).

 

(n/a) Although an analytical method was validated to measure free and total BPAF in rats and mice with good inter- and intra-day precision and accuracy. Free and total BPAF data from rats and mice were modeled using two-compartment models. While the model fit appears reason- able, the high SE values for the distribution phase reflect the rapid absorption and metabolism, with few time points available to define these parts of the curves.
Overall, these data suggest linear kinetics of free BPAF over the dose range examined and absence of sex difference in kinetics in rats. (Waidyanatha et al. 2019)

 

Excretion/Elimination: the n-Octanol/water partition coefficient (log Pow of 2.79) suggests accumulation of this substance in fatty tissues after absorption from gastro-intestinal tract is not significant, but the substance could enter circulation via lymphatic system. Based on the molecular structures and solubilities, excretion into urine as conjugated metabolites as well as parent compound in faeces, it can be assumed that the preferred route of elimination of parent is via faeces and the preferrred route of elimination of metabolites is via urine. Elimination of metabolites is assumed to be rapid as demonstrated by the declined in conjugates within 3 hours of exposure, however, some potential for bioaccumulation is to be expected, as demonstrated by the half-life of BPAF in rat i.e. about 48 hours. This may be due to the deconjugation of metabolites by the intestinal micro flora, and is demonstrated by the accumulation of the test item in the testes of exposed animals. However, the concentration of the metabolites was 30 – 40 fold more than the parent compound and furthermore, the concentration of BPAF in tissues such as the liver and kidney was over 10 fold more than compare to the concentration in testes.  The highest level of BPAF was observed in the faeces, which indicates that most of the BPAF was excreted as the nonconjugated form.  These are evidences that the potential accumulation due to deconjugation was insignificant (Li 2013, Yang 2012).

 

Supporting data and information are provided by Waidyanatha et al., 2019.

(Rats)

Total BPAF was cleared from plasma with an elimination half-life (K10 half-life) of 0.753 and 0.804 h for male and females, respectively.

Absorption of BPAF following a 34 mg/kg gavage dose in mice was more rapid than in rats with free BPAF. (see Waidyanatha et al - 2019.Mice reference RSS)

Free BPAF was cleared rapidly in mice. Similar to rats, total BPAF was much higher than corresponding free BPAF. There was no apparent sex-related effect in plasma toxicokinetic parameters of free or total BPAF in mice and rats. ((see Waidyanatha et al - 2019.Mice reference RSS)

Bioavailability in rats was with no apparent dose-related effect.

(Mice)

In this study the authors investigated the toxicokinetics and bioavailability of bisphenol AF (BPAF) in male and female B6C3F1/N mice following a single gavage administration of 34, 110, or 340 mg/kg. A validated analytical method was used to quantitate free and total BPAF in plasma.

Absorption of BPAF following a 34 mg/kg gavage dose in mice was more rapid than in rats with free BPAF. (see Waidyanatha et al - 2019.Rats reference RSS)

Free BPAF was cleared rapidly in mice. Total BPAF was much higher than corresponding free BPAF. There was no apparent sex-related effect in plasma toxicokinetic parameters of free or total BPAF in mice.

These data demonstrate that BPAF was rapidly absorbed following gavage administration in rodents, rapidly and with low bioavailability.