Reaction mass of 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol and benzyltriphenylphosphonium, salt with 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl) ethylidene]bis[phenol] (1:1)

Endocrine disrupter mammalian screening – in vivo (level 3)

Administrative data

Endpoint:

endocrine disrupter mammalian screening – in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study design is similar to pubertal assay of OECD GD 150

Data source

Reference

Reference Type:

publication

Title:

Gestational and lactational exposure to bisphenol AF in maternal rats increases testosterone levels in 23-day-old male offspring

Author:

Li et al

Year:

2016

Bibliographic source:

Chemosphere 163 (2016) 552-561

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 890.1450 (Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats

Version / remarks:

Non-guidline study in female rats similar to the pubertal assay

Deviations:

not specified

GLP compliance:

not specified

Test type:

in vivo

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

Cas Number:

1478-61-1

Molecular formula:

C15H10F6O2

IUPAC Name:

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals and environmental conditions:

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: Dissolved in corn oil
- 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 corn oil
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- 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

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Geastationday (GD) 3 to gestation day (GD) 19 and postnatal period (PD 3 to PD 190

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30

Control animals:

yes

Details on study design:

The test item dissolved in corn oil was adminsitered daily via oral gavage to femle rats from gestational day (GD) 3 to GD 19. During the gestational period, 30 females from the control group and 30 females from the treatment group were treated daily at doses of 0 or 100 mg BPAF/kg of body weight, respectively. Litters born to treated and control dams were cross-fostered, resulting in the following groups: unexposed control (CC group), pups exposed prenatally (TC group), pups exposed postnatally (CT group), and pups exposed both prenatally and postnatally (TT group). During the postnatal period (PD 3 to PD 19), cross-fostered mothers were given BPAF dissolved in corn oil orally at doses of either 0 (CC and TC) or 100 mg/kg/day BPAF (CT and TT), respectively. The mothers were weighed every 3 day and the pups were weighed every 6 day. All animals were weighed and euthanized at PD 23. Blood was collected and centrifuged at 3000 rpm at 4 C. The serum was then collected and stored at - 80 C until further analysis. Testes and epididymides were isolated and weighed. Testes were frozen in liquid nitrogen and stored at - 80 C for RNA isolation and protein extraction.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of the GD 9 to GD 18 treatment group mothers and pups showed significant decrease.

Food consumption and compound intake (if feeding study):

not specified

Water consumption and compound intake (if drinking water study):

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in testis testosterone levels

Significant decrease in testis inhibin B (INHB) levels

279 genes were significantly differentially expressed in the testes of pups exposed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Any other information on results incl. tables

BPAF concentration in serum and testis

The concentrations of free and total BPAF in serum and testis were detected in all groups via LC-MS/MS. The concentrations of free BPAF in serum were 0.21, 0.34, 9.32, and 29.38 ng/mL, and the concentrations of total BPAF were 0.76, 1.32, 27.60, and 127.00 ng/mL in the CC, TC, CT, and TT groups, respectively. Free andt otal BPAF levels in serum were ranked as TT group > CT group > TC group > CC group. The content of free and total BPAF in the TC and CT groups indicated that BPAF was transferred to infants via gestation and lactation, although the concentration of BPAF in cord blood and breast milk was not measured directly. Furthermore, BPAF in the serumwas more easily eliminated than that in the testis during exposure.

Hormone levels in serum and testis

No significant differences were observed in the levels of serum estradiol, luteinizing hormone, or follicle-stimulating hormone among the four groups, except for a decrease in the TC and CT group compared with the TT group. Serum anti-Müllerian hormone (AMH) levels in the TT group were lower than those of the CC, TC and CT group, respectively (p < 0.05). Only the TT group serum and testis testosterone levels showed significant increase compared with those of the CC group (p < 0.05, Fig. 2). The levels of INHB in the serum of the TC and TT groups decreased compared with those of the CC group (p < 0.05), while the content of INHB in the testis of all treated groups decreased significantly compared with that of the control group (p < 0.01).

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

In this non-guideline test, female rats from gestational day (GD) 3-19 were exposed to 100 mg BPAF/kg/day by oral gavage. On the day of birth (postnatal day (PD) 0), cross-fostering took place between treated and control litters, and cross-fostered mother rats were given BPAF 100 mg/kg/day during the postnatal period (PD 3 to PD 19).

HPLC-MS/MS analysis showed that BPAF was transferred via cord blood and lactation, finally bioaccumulating in the offspring testes.

Body weights of the GD 9 to GD 18 treatment group mothers showed significant decrease.

No effect on the absolute and relative weights of the testis and the absolute weights of the epididymis in pups.

Free and total BPAF levels in serum was high in all treatment group i.e. material and pup, indicating that BPAF was transferred to infants via gestation and lactation.  Free and total BPAF levels in testis was detected in all treatment groups.  Testis testosterone levels showed significant increase in pups exposed to pre/postnatal and there was 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. The protein levels of genes involved in steroidogenesis (P450scc and StAR) in the male rat testis increased in pre/post-natal exposure group, including genes involved in cell differentiation and meiosis.

These results indicate that gestational and loctational exposure to BPAF in the mother can impair reproductive function in male offspring.