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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.

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.

Description of key information

In the two toxicokinetics studies (Yang et al. 2012, Li et al. 2013) (conducted on BPAF) the following was observed:

There was no absorption data 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.

There are 2 endocrine disruption screening - in vivo studies, (conducted on BPAF), entered in IUCLID section 7.9.3 Endocrine disrupter mammalian screening, which indicate the folowing:

- Li et al 2016 :

Pups exposed to BPAF both prenatally and postnatally showed a significant increase in testis testosterone levels compared with that of the control, while all pups exposed to BPAF showed a significant decrease in testis inhibin B (INHB) levels. Compared with the control, RNA-seq revealed that 279 genes were significantly differentially expressed in the testes of pups exposed to BPAF both prenatally and postnatally, including genes involved in cell differentiation and meiosis. These results indicate that gestational and lactational exposure to BPAF in the mother can impair reproductive function in male offspring.This test was not conducted in accordance with International guidance and has significant methological deficincies when compared to OECD reproductive toxicity tests.

- Yamasaki et al 2003:

The NOAEL for body weight increase in female and male rats was 100 and 50 mg/kg bw per day, respectively.  The NOAEL for mortality in male rats was 200 mg/kg bw per day, where mortality was observed in the top concentration (600 mg/kg bw per day).In the uterotrophic test, a significant increase in relative uterus weight (p < 0.01) was observed in female rats treated at 8 and 40 mg/kg bw per day. In the Hershberger test, a significant reduction (p < 0.05) in BC/LA weight and a significant increase (p < 0.05) in glans penis weight was observed in male rats treated at 200 and 600 mg/kg bw per day, respectively.

There are the following 3 pieces of information (conducted on BPAF) entered in IUCLID section 7.9.4 - Specific Investigations:

1 - Li et al. 2013 (endocrine Disruption) in vivo WOE, which examines the agonistic activity of bisphenol-AF through ERα and ERβ and investigated the effects of bisphenol-AF on ER-mediated target genes. The conclusions were Bisphenol-AF strongly activated ERα estrogen responsive element (ERE)-mediated responses. Results of real-time polymerase chain reaction indicated that bisphenol AF consistently activated endogenous ER target genes.

2 - Matsushima et al 2010 (endocrine disruption) in vivo WOE, which determines the relative preference of bisphenol AF for the human nuclear estrogenic receptors ERα and ERβ and the bisphenol A–specific estrogen-related receptor ERRγ, and clarifies structural characteristics of receptors that influence bisphenol AF binding. It concluded Bisphenol AF could function as an endocrine-disrupting chemical by acting as an agonist or antagonist to perturb physiological processes mediated through ERα and/or ERβ.

3 - Li et al 2012 (endocrine disruption) In vitro models were used to evaluate the mechanistic actions of BPAF on estrogen receptor (ER) α and ERβ. which concludes that BPAF can function as an endocrine disrupting chemical by acting as an agonist (at higher concentrations; ≥ 10 nM) and as an antagonists (at lower concentrations; ≤ 10 nM) for ERα and ERβ. These actions were found to be cell-type specific. BPAF activated the AF‑2 function of ERα in addition to inducing the p44/42 MAPK pathway, suggesting that it can mediate rapid action responses involved in endogenous ER signaling events as well as genomic responses. Taken together, the data demonstrate the mechanistic importance of cell type specificity in evaluation of the potential activities of BPAF.

Overall conclusion for BPAF reaction mass:

There is a significant amount of data on BPAF toxicokinetics. Because BPAF is a major component of the reaction mass, and because the observed toxicity of the reaction mass appears to be consistent with BPAF exposure, the information on BPAF toxicokinetics is included here.

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 BPAF (Yang 2012).

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).

 

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).