Phenol, mono- + distyrenated (MSP+DSP)

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

endocrine system modulation

Remarks:

in vitro Transcriptional Activation assays

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: To assess the potential endocrine disrupting properties of MSP, we developed an in vitro strategy as recommended in the OECD Conceptual Framework and the US EPA EDPS for the Testing and Assessment of ED chemicals (Series on testing and assessment n° 118 - 18-Jan-2012), using immortalized cell lines expressing various nuclear receptors that could be putative targets for TSP and its potential impurities or components (mono -MSP- and di-styrenated -DSP- phenol) and that could mediate endocrine disruption.
- Short description of test conditions:
The two isomers of MSP (2-MSP and 4-MSP) and the mixture of 2-MSP and 4-MSP was tested on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: to assess the potential endocrine disrupting properties of MSP

Specific details on test material used for the study:

Chemicals sent by Solvay were dissolved in DMSO at 10-2 M for storage at -20°C.
The two isomers of MSP (2-MSP and 4-MSP) and the mixture of 2-MSP and 4-MSP was tested on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.

Details on study design:

Cell lines
All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line
HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob-Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line
HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).
Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4-chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4-PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions
HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013).

Test conditions
All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% desteroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.
Cells were plated 24h at 20000 cells/well (200 μL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours.
After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.
These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α and hER-β, antagonism properties were tested in the presence of 0.1 nM E2.

References:
Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Casellas, C., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47-56.

Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al. (2012). Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012). Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies. Toxicological Sciences, 125(2), 359-367.

Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Examinations:

Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

Details on results:

Non-specific effects
Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).
On HELN cell line, MSP, 2-MSP, 4-MSP did not present any non-specific activity up 10 µM.
In the following tests, the maximum concentration tested was set at 33 µM to avoid general cytotoxicity.
On HG5LN cell line, at 33 µM, MSP showed a slight non-specific activity.
In the following tests, the maximum concentration tested was set at 10 µM to avoid general cytotoxicity and effects occurring at 3 and 10 µM would be considered as non-specific effects.

Estrogen receptors activity
Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).
In HELN ERα cells, the mix MSP presented a dose-related ERα agonist activity which was evident at 3, 10 µM and 33 µM. In HELN ERβ cells, only MSP showed a significant agonist activity starting from 1 µM. Calculated EC50s for the mix MSP on ERα and ERβ were respectively 5.7 and 5.1 µM. In 2016 new experiments were performed in order to identify which MSP isomer was responsible for the estrogenic activity. It appears clearly that 4-MSP is the isomer bearing the estrogenic activity of the mix MSP. EC50 for 2-MSP and 4-MSP on ERα were 38.2 and 3.2 µM respectively, EC50 for 2-MSP and 4-MSP on ERβ were 16.0 and 1.4 µM respectively.

Conclusions:

MSP had agonist properties on estradiol receptors (hERα and hERβ), 4-MSP being much more potent than 2-MSP on both hERα and hERβ.
Mix MSP EC5O values for ERα and ERβ (5.7 and 5.1 µM respectively) are comprised in between those of 2-MSP (38.2 and 16 µM respectively) and 4-MSP (3.2 and 1.4 µM respectively), thus suggesting that the estrogenic activity of the mix MSP is mostly carried by the 4-MSP isomer.
MSP presented a significant estrogenic activity on both human estrogen receptors, but being 300 000 times less potent than estradiol, 20 times less potent than nonylphenol and 10 times less potent than bisphenol-A (BPA).

Executive summary:

Many endocrine disruptors exert their harmful effects by interacting with nuclear receptors, which in turn leads to deregulation of gene transcription. Among these nuclear receptors are hormone receptors (glucocorticoid, mineralocorticoid, oestrogen, progestagen and androgens receptors). Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Hormone nuclear receptors, such as thyroid hormone receptors (TRs) and oestrogen receptors (ERs), are well conserved across evolution, especially in mammals. Therefore results obtained with transactivation assays in vitro with a species specific receptor are transposable to other species.

Other nuclear receptors, called xenosensors, are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. These receptors are also involved in hormones turnover and half-life and their activation or repression participate to general endocrine disruption. They are less conserved across evolution and results obtained in transactivation studies are not always transposable to receptors from other species. These molecular variations are the basis of differences observed across species in xenobiotic metabolism.

The aim of the present study was to assess the potential endocrine disrupting properties of tristyrenated phenol (TSP) and its potential impurities or components (mono -MSP- and distyrenated-DSP- phenol).

An in vitro strategy was developed using immortalized luciferase-reporter cell lines expressing various nuclear receptors that could be putative targets for TSP and that could mediate endocrine disruption.

The two isomers of MSP (2-MSP and 4-MSP) and the mixture of 2-MSP and 4-MSP was tested on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.

Cell lines

All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line

HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line

HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).

Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4-chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4-PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions

HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013).

Test conditions

All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% desteroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.

Cells were plated 24h at 20000 cells/well (200 μL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours.

After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.

These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α and hER-β, antagonism properties were tested in the presence of 0.1 nM E2.

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity

(DMSO).

On HELN cell line, MSP, 2-MSP, 4-MSP did not present any non-specific activity up 10 μM.

In the following tests, the maximum concentration tested was set at 33 μM to avoid general cytotoxicity.

On HG5LN cell line, at 33 μM, MSP showed a slight non-specific activity.

In the following tests, the maximum concentration tested was set at 10 μM to avoid general cytotoxicity and effects occurring at 3 and 10 μM would be considered as non-specific effects.

Estrogen receptors activity

Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).

In HELN ERα cells, the mix MSP presented a dose-related ERα agonist activity which was evident at 3, 10 μM and 33 μM. In HELN ERβ cells, only MSP showed a significant agonist activity starting from 1 μM. Calculated EC50s for the mix MSP on ERα and ERβ were respectively 5.7 and 5.1 μM. In 2016 new experiments were performed in order to identify which MSP isomer was responsible for the estrogenic activity. It appears clearly that 4-MSP is the isomer bearing the estrogenic activity of the mix MSP. EC50 for 2-MSP and 4-MSP on ERα were 38.2 and 3.2 μM respectively, EC50 for 2-MSP and 4-MSP on ERβ were 16.0 and 1.4 μM respectively.

Conclusions

MSP had agonist properties on estradiol receptors (hERα and hERβ), 4-MSP being much more potent than 2-MSP on both hERα and hERβ.

Mix MSP EC5O values for ERα and ERβ (5.7 and 5.1 μM respectively) are comprised in between those of 2-MSP (38.2 and 16 μM respectively) and 4-MSP (3.2 and 1.4 μM respectively), thus suggesting that the estrogenic activity of the mix MSP is mostly carried by the 4-MSP isomer.

MSP presented a significant estrogenic activity on both human estrogen receptors, but being 300 000 times less potent than estradiol, 20 times less potent than nonylphenol and 10 times less potent than bisphenol-A (BPA).

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Case llas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al.(2012). Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

- Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012). Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies.Toxicological Sciences, 125(2), 359-367.

- Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Reference 2

Endpoint:

endocrine system modulation

Remarks:

in vitro Transcriptional Activation assays

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: To assess the potential endocrine disrupting properties of 2,4-DSP, we developed an in vitro strategy as recommended in the OECD Conceptual Framework and the US EPA EDPS for the Testing and Assessment of ED chemicals (Series on testing and assessment n° 118 - 18-Jan-2012), using immortalized cell lines expressing various nuclear receptors that could be putative targets for TSP and its potential impurities or components (mono -MSP- and di-styrenated -DSP- phenol) and that could mediate endocrine disruption.
- Short description of test conditions:
2,4-DSP was tested on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.
2,4-DSP was tested in vitro on immortalized cell lines on rat thyroid hormones receptors (rTRα, rTRβ).
2,4-DSP was tested in vitro on immortalized cell lines on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: to assess the potential endocrine disrupting properties of 2,4-DSP

Specific details on test material used for the study:

Chemicals sent by Solvay were dissolved in DMSO at 10-2 M for storage at -20°C.

Details on study design:

Cell lines
All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line
HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob-Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line
HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).
Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4- chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4-PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions
HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013). HG5LN GAL4-hPXR, -mPXR, -mCAR, -rTR-α and -rTR-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red, 1g/L glucose and supplemented with 5% de-steroidated fetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Fini et al., 2012, Ménez et al., 2012).

Test conditions
All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% de-steroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.
Cells were plated 24h at 20000 cells/well (200 µL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours. After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.
These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α, hER-β, rTR-α and rTR-β, antagonism properties were tested in the presence of 0.1 nM E2 for hER-α, hER-β, and 1nM T3 for rTR-α and rTR-β.

References:
Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Casellas, C., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47-56.

Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al. (2012).
Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012).
Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies. Toxicological Sciences, 125(2), 359-367.

Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M., & Lespine, A.
(2012). Ivermectin induces P-glycoprotein expression and function through mRNA stabilization
in murine hepatocyte cell line. Biochemical Pharmacology, 83(2), 269-278.

Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Examinations:

Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

Details on results:

Non-specific effects
Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).
On HELN cell line, 2,4-DSP did not present any non-specific activity up to 10 μM.
A general toxicity was observed for concentrations above 33 μM for 2,4-DSP.
In the following tests, the maximum concentration tested was set at 33 μM to avoid general cytotoxicity.
On HG5LN cell line, 2,4-DSP showed clear non-specific activity at 10 μM and a tendency for non-specific activity at 3 μM. At 33 μM, 2,4-DSP showed a general cytotoxic effect assessed by a decrease in luciferase activity relative to control (vehicle DMSO).
In the following tests, the maximum concentration tested was set at 10 μM to avoid general cytotoxicity and effects occurring at 3 and 10 μM would be considered as non-specific effects.

Estrogen receptors activity
Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).
In HELN ERα cells, 2,4-DSP did not show any significant agonist activity. In HELN ERβ cells, 2,4-DSP did not show any significant agonist activity.

Thyroid hormone receptors activity
Agonistic activity
Luciferase activity was scaled as percentage of the luciferase activity induced by 100 nm T3. 2,4-DSP did not (range 10 nM-10 μM) induce luciferase activity mediated through TRs transactivation.

Antagonistic activity
Antagonistic activity was assessed in the presence of 1 nM T3, a value close to EC50, and expressed as percentage relative to luciferase activity measured in the presence of 100 nM T3 (100 nM T3 provided the maximal transactivation of TRs). An antagonist activity should appear as a downward inflexion of the dose response curve. In HG5LN rTRα cells, 2,4-DSP slightly increased luciferase activity at 3 and 10 μM, here again in the range of non-specific effects seen in HG5LN. In HGSLN rTRβ cells, no antagonistic activity was seen with 2,4-DSP. A slight increase in luciferase activity was observed with 2,4-DSP at 10 μM but this was a concentration at which non-specific effects were seen.

Pregnane X receptor activity
PXR transactivation was measured relative to the effect of the hPXR agonist 10 μM SR12813 (SR).
2,4-DSP presented a specific PXR activity. EC50 estimated for 2,4-DSP were 3.67 μM for hPXR, and 2.31 μM for mPXR.

Constitutive androstane receptor activity
hCAR has a high constitutive activity. To show an antagonistic activity, molecules can be directly tested on the HG5LN-hCAR cell line. To detect any putative agonistic activity, constitutive hCAR activity was blocked by the inverse agonist 3 μM PK1195 (IC50=0.71 μM). None of the three molecules showed specific agonistic or antagonistic activity. The apparent increases in hCAR activity seen at 10μM are considered to be the result of non-specific effects.
As for its human counterpart, mCAR presents a high constitutive activity, still being stimulated by the agonist TCPOBOP. To enhance the detection of an agonistic activity the inverse agonist T0901317 was used (IC50 1.54 μM). 2,4-DSP presented an agonistic activity and was able to increase mCAR activity with an EC50 value of 1.1 μM. In the presence of the inverse agonist T0901317 at 10 μM, 2,4-DSP showed an agonist activity with an EC50 value of 4.01 μM.

Conclusions:

2,4-DSP displayed no agonist properties on estradiol receptors.
2,4-DSP presented no specific activities on rat thyroid hormones receptors. TRs are highly conserved across mammals, thus we can extrapolate these results to human TRs.
Regarding xenosensors, 2,4-DSP was found to induce transactivation of xenosensors mPXR, hPXR and mCAR with EC50 values in the micromolar range (agonist properties similar to cypermethrin).
Overall, these in vitro transactivation studies support the hypothesis that 2,4-DSP could induce an increase in thyroid hormones turnover in rats due to specificities in rodent endocrinology. Such impact on human thyroid axis is unlikely to occur.

Executive summary:

Many endocrine disruptors exert their harmful effects by interacting with nuclear receptors, which in turn leads to deregulation of gene transcription. Among these nuclear receptors are hormone receptors (glucocorticoid, mineralocorticoid, oestrogen, progestagen and androgens receptors). Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Hormone nuclear receptors, such as thyroid hormone receptors (TRs) and oestrogen receptors (ERs), are well conserved across evolution, especially in mammals. Therefore results obtained with transactivation assays in vitro with a species specific receptor are transposable to other species.

Other nuclear receptors, called xenosensors, are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. These receptors are also involved in hormones turnover and half-life and their activation or repression participate to general endocrine disruption. They are less conserved across evolution and results obtained in transactivation studies are not always transposable to receptors from other species. These molecular variations are the basis of differences observed across species in xenobiotic metabolism.

The aim of the present study was to assess the potential endocrine disrupting properties of tristyrenated phenol (TSP) and its potential impurities or components (mono -MSP- and distyrenated-DSP- phenol).

An in vitro strategy was developed using immortalized luciferase-reporter cell lines expressing various nuclear receptors that could be putative targets for TSP and that could mediate endocrine disruption.

2,4-DSP was tested in vitro:

- on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines;

- on immortalized cell lines on rat thyroid hormones receptors (rTRα, rTRβ);

- on immortalized cell lines on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination.

Cell lines

All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line

HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line

HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).

Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4-chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4 -PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions

HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013). HG5LN GAL4-hPXR, -mPXR, -mCAR, -rTR-α and -rTR-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red, 1g/L glucose and supplemented with 5% de-steroidated fetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Fini et al., 2012, Ménez et al., 2012).

Test conditions

All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% desteroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.

Cells were plated 24h at 20000 cells/well (200 μL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours.

After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.

These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α, hER-β, rTR-α and rTR-β, antagonism properties were tested in the presence of 0.1 nM E2 for hER-α, hER-β, and 1nM T3 for rTR-α and rTR-β.

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).

On HELN cell line, 2,4-DSP did not present any non-specific activity up 10 μM.

A general toxicity was observed for concentrations above 33 μM for 2,4-DSP (<TSP<2,6-DSP).

In the following tests, the maximum concentration tested was set at 33 μM to avoid general cytotoxicity.

On HG5LN cell line, 2,4-DSP showed clear non-specific activity at 10 μM and a tendency for nonspecific activity at 3 μM. At 33 μM, 2,4-DSP showed a general cytotoxic effect assessed by a decrease in luciferase activity relative to control (vehicle DMSO).

In the following tests, the maximum concentration tested was set at 10 μM to avoid general cytotoxicity and effects occurring at 3 and 10 μM would be considered as non-specific effects.

Estrogen receptors activity

Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).

In HELN ERα cells, 2,4-DSP did not show any significant ERα agonist activity. In HELN ERβ cells, only MSP showed a significant agonist activity starting from 1 μM.

Thyroid hormone receptors activity

Agonistic activity

Luciferase activity was scaled as percentage of the luciferase activity induced by 100 nm T3. None of the three molecules tested (range 10 nM-10 μM) induced luciferase activity mediated through TRs

transactivation.

Antagonistic activity

Antagonistic activity was assessed in the presence of 1 nM T3, a value close to EC50, and expressed as percentage relative to luciferase activity measured in the presence of 100 nM T3 (100 nM T3 provided the maximal transactivation of TRs). An antagonist activity should appear as a downward inflexion of the dose response curve. In HG5LN rTRα cells, 2,4-DSP slightly increased luciferase activity at 3 and 10 μM, here again in the range of non-specific effects seen in HG5LN. In HGSLN rTRβ cells, no antagonistic activity was seen with 2,4-DSP. A slight increase in luciferase activity was observed with 2,4- DSP at 10 μM but this was a concentration at which non-specific effects were seen.

Pregnane X receptor activity

PXR transactivation was measured relative to the effect of the hPXR agonist 10 μM SR12813 (SR).

2,4-DSP presented a specific PXR activity: TSP>2,6-DSP>2,4-DSP. EC50 estimated for 2,4,6 TSP, 2,6-DSP and 2,4-DSP were respectively 1.71, 2.3 and 3.67 μM for hPXR, and 1.07, 1.88 and 2.31 μM for mPXR.

Constitutive androstane receptor activity

hCAR has a high constitutive activity. To show an antagonistic activity, molecules can be directly tested on the HG5LN-hCAR cell line. To detect any putative agonistic activity, constitutive hCAR activity was blocked by the inverse agonist 3 μM PK1195 (IC50=0.71 μM). None of the three molecules showed specific agonistic or antagonistic activity. The apparent increases in hCAR activity seen at 10 μM are considered to be the result of non-specific effects.

As for its human counterpart, mCAR presents a high constitutive activity, still being stimulated by the agonist TCPOBOP. To enhance the detection of an agonistic activity the inverse agonist T0901317 was used (IC50 1.54 μM). The three molecules 2,4,6-TSP, 2,4-DSP and 2,6-DSP presented an agonistic activity and were able to increase mCAR activity: 2,4 DSP > 2,6 DSP > 2,4,6 TSP with respective EC50 values: 1.1, 1.44 and 4.77 μM. In the presence of the inverse agonist T0901317 at 10 μM, all three molecules showed an agonist activity: 2,6 DSP = 2,4,6 TSP > 2,4 DSP with respective EC50 values: 2.65, 3.13 and 4.01 μM.

Conclusions

2,4-DSP displayed no agonist properties on estradiol receptors.

2,4-DSP presented no specific activities on rat thyroid hormones receptors. TRs are highly conserved across mammals, thus we can extrapolate these results to human TRs.

Regarding xenosensors, 2,4-DSP was found to induce transactivation of xenosensors mPXR, hPXR and mCAR with EC50 values in the micromolar range (agonist properties similar to cypermethrin).

Overall, these in vitro transactivation studies support the hypothesis that 2,4-DSP could induce an increase in thyroid hormones turnover in rats due to specificities in rodent endocrinology. Such impact on human thyroid axis is unlikely to occur.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Case llas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al.(2012). Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

- Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012). Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies.Toxicological Sciences, 125(2), 359-367.

- Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M., & Lespine, A. (2012).Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line. Biochemical Pharmacology, 83(2), 269-278.

- Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Reference 3

Endpoint:

endocrine system modulation

Remarks:

in vitro Transcriptional Activation assays

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: To assess the potential endocrine disrupting properties of 2,6-DSP, we developed an in vitro strategy as recommended in the OECD Conceptual Framework and the US EPA EDPS for the Testing and Assessment of ED chemicals (Series on testing and assessment n° 118 - 18-Jan-2012), using immortalized cell lines expressing various nuclear receptors that could be putative targets for TSP and its potential impurities or components (mono -MSP- and di-styrenated -DSP- phenol) and that could mediate endocrine disruption.
- Short description of test conditions:
2,6-DSP was tested on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.
2,6-DSP was tested in vitro on immortalized cell lines on rat thyroid hormones receptors (rTRα, rTRβ).
2,6-DSP was tested tested in vitro on immortalized cell lines on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: to assess the potential endocrine disrupting properties of 2,6-DSP

Specific details on test material used for the study:

Chemicals sent by Solvay were dissolved in DMSO at 10-2 M for storage at -20°C.

Details on study design:

Cell lines
All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line
HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob-Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line
HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).
Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4-chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4-PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions
HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013). HG5LN GAL4-hPXR, -mPXR, -mCAR, -rTR-α and -rTR-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red, 1g/L glucose and supplemented with 5% de-steroidated fetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Fini et al., 2012, Ménez et al., 2012).

Test conditions
All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% desteroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.
Cells were plated 24h at 20000 cells/well (200 μL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours.
After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.
These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α, hER-β, rTR-α and rTR-β, antagonism properties were tested in the presence of 0.1 nM E2 for hER-α, hER-β, and 1nM T3 for rTR-α and rTR-β.

References:
Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Casellas, C., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47-56.

Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al. (2012). Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.
Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012). Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies. Toxicological Sciences, 125(2), 359-367.

Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M., & Lespine, A. (2012). Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line. Biochemical Pharmacology, 83(2), 269-278.

Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Examinations:

Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

Details on results:

Non-specific effects
Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).
On HELN cell line, 2,6-DSP at 10 µM presented low to high non-specific activation of luciferase. A general toxicity was observed for concentrations above 33 µM for 2,6-DSP.
In the following tests, the maximum concentration tested was set at 33 µM to avoid general cytotoxicity and effects occurring at 10 µM and above for 2,6-DSP would be considered as non-specific effects.
On HG5LN cell line, 2,6-DSP showed clear non-specific activity at 10 µM and a tendency for non-specific activity at 3 µM. At 33 µM, 2,6-DSP showed a general cytotoxic effect assessed by a decrease in luciferase activity relative to control (vehicle DMSO).
In the following tests, the maximum concentration tested was set at 10 µM to avoid general cytotoxicity and effects occurring at 3 and 10 µM would be considered as non-specific effects.

Estrogen receptors activity
Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).
In HELN ERα cells, 2,6-DSP presented an apparent agonist activity on ERα but this was obtained at the concentration of 10 µM known to induce non·specific effects. In HELN ERβ cells, 2,6-DSP did not show any significant agonist activity.

Thyroid hormone receptors activity
Agonistic activity
Luciferase activity was scaled as percentage of the luciferase activity induced by 100 nm T3. 2,6-DSP (range 10 nM-10 µM) did not induce luciferase activity mediated through TRs transactivation.

Antagonistic activity
Antagonistic activity was assessed in the presence of 1 nM T3, a value close to EC50, and expressed as percentage relative to luciferase activity measured in the presence of 100 nM T3 (100 nM T3 provided the maximal transactivation of TRs). An antagonist activity should appear as a downward inflexion of the dose response curve. In HG5LN rTRα cells, 2,6-DSP slightly increased luciferase activity at 3 and 10 µM, here again in the range of non-specific effects seen in HG5LN. In HGSLN rTRβ cells, no antagonistic activity was seen with 2,6-DSP. A slight increase in luciferase activity was observed with 2,6-DSP at 10 µM but this was a concentration at which non-specific effects were seen.

Pregnane X receptor activity
PXR transactivation was measured relative to the effect of the hPXR agonist 10 µM SR12813 (SR). 2,6-DSP presented a specific PXR activity. EC50 estimated for 2,6-DSP were 2.3 µM for hPXR, and 1.88 µM for mPXR.

Constitutive androstane receptor activity
hCAR has a high constitutive activity. To show an antagonistic activity, molecules can be directly tested on the HG5LN-hCAR cell line. To detect any putative agonistic activity, constitutive hCAR activity was blocked by the inverse agonist 3 µM PK1195 (IC50=0.71 µM). None of the three molecules showed specific agonistic or antagonistic activity. The apparent increases in hCAR activity seen at 10 µM are considered to be the result of non-specific effects.
As for its human counterpart, mCAR presents a high constitutive activity, still being stimulated by the agonist TCPOBOP. To enhance the detection of an agonistic activity the inverse agonist T0901317 was used (IC50 1.54 µM). 2,6-DSP presented an agonistic activity and was able to increase mCAR activity, with an EC50 value of 1.44 µM. In the presence of the inverse agonist T0901317 at 10 µM, 2,6-DSP showed an agonist activity, with an EC50 value of 2.65 µM.

Conclusions:

2,6-DSP displayed no agonist properties on estradiol receptors.
2,6-DSP presented no specific activities on rat thyroid hormones receptors. TRs are highly conserved across mammals, thus we can extrapolate these results to human TRs.
Regarding xenosensors, 2,6-DSP was found to induce transactivation of xenosensors mPXR, hPXR and mCAR with EC50 values in the micromolar range (agonist properties similar to cypermethrin).
Overall, these in vitro transactivation studies support the hypothesis that 2,6-DSP could induce an increase in thyroid hormones turnover in rats due to specificities in rodent endocrinology. Such impact on human thyroid axis is unlikely to occur

Executive summary:

Many endocrine disruptors exert their harmful effects by interacting with nuclear receptors, which in turn leads to deregulation of gene transcription. Among these nuclear receptors are hormone receptors (glucocorticoid, mineralocorticoid, oestrogen, progestagen and androgens receptors). Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Hormone nuclear receptors, such as thyroid hormone receptors (TRs) and oestrogen receptors (ERs), are well conserved across evolution, especially in mammals. Therefore results obtained with transactivation assays in vitro with a species specific receptor are transposable to other species.

Other nuclear receptors, called xenosensors, are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. These receptors are also involved in hormones turnover and half-life and their activation or repression participate to general endocrine disruption. They are less conserved across evolution and results obtained in transactivation studies are not always transposable to receptors from other species. These molecular variations are the basis of differences observed across species in xenobiotic metabolism.

The aim of the present study was to assess the potential endocrine disrupting properties of tristyrenated phenol (TSP) and its potential impurities or components (mono -MSP- and distyrenated-DSP- phenol).

An in vitro strategy was developed using immortalized luciferase-reporter cell lines expressing various nuclear receptors that could be putative targets for TSP and that could mediate endocrine disruption.

2,6-DSP was tested in vitro:

- on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.

- on immortalized cell lines on rat thyroid hormones receptors (rTRα, rTRβ).

- on immortalized cell lines on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination.

Cell lines

All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line

HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line

HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).

Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4-chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4-PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions

HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013). HG5LN GAL4-hPXR, -mPXR, -mCAR, -rTR-α and -rTR-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red, 1g/L glucose and supplemented with 5% de-steroidated fetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Fini et al., 2012, Ménez et al., 2012).

Test conditions

All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% desteroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.

Cells were plated 24h at 20000 cells/well (200 μL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours.

After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.

These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α, hER-β, rTR-α and rTR-β, antagonism properties were tested in the presence of 0.1 nM E2 for hER-α, hER-β, and 1nM T3 for rTR-α and rTR-β.

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).

On HELN cell line, 2,6-DSP at 10 μM presented low to high non-specific activation of luciferase.

A general toxicity was observed for concentrations above 33 μM for TSP and DSPs (2,4-DSP<TSP<2,6-DSP).

In the following tests, the maximum concentration tested was set at 33 μM to avoid general cytotoxicity and effects occurring at 10 μM and above for 2,6-DSP would be considered as non-specific effects.

On HG5LN cell line, 2,6-DSP showed clear non-specific activity at 10 μM and a tendency for non-specific activity at 3 μM. At 33 μM, 2,6-DSP showed a general cytotoxic effect assessed by a decrease in luciferase activity relative to control (vehicle DMSO).

In the following tests, the maximum concentration tested was set at 10 μM to avoid general cytotoxicity and effects occurring at 3 and 10 μM would be considered as non-specific effects.

Estrogen receptors activity

Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).

In HELN ERα cells, 2,6-DSP presented an apparent agonist activity on ERα but this was obtained at the concentration of 10 μM known to induce non·specific effects. In HELN ERβ cells, only MSP showed a significant agonist activity starting from 1 μM.

Thyroid hormone receptors activity

Agonistic activity

Luciferase activity was scaled as percentage of the luciferase activity induced by 100 nm T3. None of the three molecules tested (range 10 nM-10 μM) induced luciferase activity mediated through TRs transactivation.

Antagonistic activity

Antagonistic activity was assessed in the presence of 1 nM T3, a value close to EC50, and expressed as percentage relative to luciferase activity measured in the presence of 100 nM T3 (100 nM T3 provided the maximal transactivation of TRs). An antagonist activity should appear as a downward inflexion of the dose response curve. In HG5LN rTRα cells, 2,6-DSP slightly increased luciferase activity at 3 and 10 μM, here again in the range of non-specific effects seen in HG5LN. In HGSLN rTRβ cells, no antagonistic activity was seen with 2,6-DSP. A slight increase in luciferase activity was observed with 2,6-DSP at 10 μM but this was a concentration at which non-specific effects were seen.

Pregnane X receptor activity

PXR transactivation was measured relative to the effect of the hPXR agonist 10 μM SR12813 (SR) The three molecules tested 2,4,6-TSP, 2,4-DSP, 2,6-DSP presented a specific PXR activity: TSP>2,6-DSP>2,4-DSP. EC50 estimated for 2,4,6 TSP, 2,6-DSP and 2,4-DSP were respectively 1.71, 2.3 and 3.67 μM for hPXR (Figure 5A), and 1.07, 1.88 and 2.31 μM for mPXR.

Constitutive androstane receptor activity

hCAR has a high constitutive activity. To show an antagonistic activity, molecules can be directly tested on the HG5LN-hCAR cell line. To detect any putative agonistic activity, constitutive hCAR activity was blocked by the inverse agonist 3 μM PK1195 (IC50=0.71 μM). None of the three molecules showed specific agonistic or antagonistic activity. The apparent increases in hCAR activity seen at 10 μM are considered to be the result of non-specific effects.

As for its human counterpart, mCAR presents a high constitutive activity, still being stimulated by the agonist TCPOBOP. To enhance the detection of an agonistic activity the inverse agonist T0901317 was used (IC50 1.54 μM). The three molecules 2,4,6-TSP, 2,4-DSP and 2,6-DSP presented an agonistic activity and were able to increase mCAR activity: 2,4 DSP > 2,6 DSP > 2,4,6 TSP with respective EC50 values: 1.1, 1.44 and 4.77 μM. In the presence of the inverse agonist T0901317 at 10 μM, all three molecules showed an agonist activity: 2,6 DSP = 2,4,6 TSP > 2,4 DSP with respective EC50 values: 2.65, 3.13 and 4.01 μM.

Conclusions

2,6-DSP displayed no agonist properties on estradiol receptors.

2,6-DSP presented no specific activities on rat thyroid hormones receptors. TRs are highly conserved across mammals, thus we can extrapolate these results to human TRs.

Regarding xenosensors, 2,6-DSP was found to induce transactivation of xenosensors mPXR, hPXR and mCAR with EC50 values in the micromolar range (agonist properties similar to cypermethrin).

Overall, these in vitro transactivation studies support the hypothesis that 2,6-DSP could induce an increase in thyroid hormones turnover in rats due to specificities in rodent endocrinology. Such impact on human thyroid axis is unlikely to occur.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Case llas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al.(2012). Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

- Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012). Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies.Toxicological Sciences, 125(2), 359-367.

- Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M., & Lespine, A. (2012).Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line. Biochemical Pharmacology, 83(2), 269-278.

- Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Reference 4

Endpoint:

endocrine system modulation

Remarks:

in vitro Transcriptional Activation assays

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: To assess the potential endocrine disrupting properties of TSP, we developed an in vitro strategy as recommended in the OECD Conceptual Framework and the US EPA EDPS for the Testing and Assessment of ED chemicals (Series on testing and assessment n° 118 - 18-Jan-2012), using immortalized cell lines expressing various nuclear receptors that could be putative targets for TSP and its potential impurities or components (mono -MSP- and di-styrenated -DSP- phenol) and that could mediate endocrine disruption.
- Short description of test conditions:
2,4,6-TSP was tested on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines.
2,4,6-TSP was tested in vitro on immortalized cell lines on rat thyroid hormones receptors (rTRα, rTRβ).
2,4,6-TSP was tested in vitro on immortalized cell lines on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: to assess the potential endocrine disrupting properties of TSP

Specific details on test material used for the study:

Chemicals sent by Solvay were dissolved in DMSO at 10E-2 M for storage at -20°C.

Details on study design:

Cell lines
All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line
HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob-Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line
HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).
Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4- chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4-PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions
HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013). HG5LN GAL4-hPXR, -mPXR, -mCAR, -rTR-α and -rTR-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red, 1g/L glucose and supplemented with 5% de-steroidated fetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Fini et al., 2012, Ménez et al., 2012).

Test conditions
All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% de-steroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.
Cells were plated 24h at 20000 cells/well (200 µL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours. After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.
These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α, hER-β, rTR-α and rTR-β, antagonism properties were tested in the presence of 0.1 nM E2 for hER-α, hER-β, and 1nM T3 for rTR-α and rTR-β.

References:
Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Casellas, C., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47-56.

Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al. (2012).
Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012).
Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies. Toxicological Sciences, 125(2), 359-367.

Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M., & Lespine, A.
(2012). Ivermectin induces P-glycoprotein expression and function through mRNA stabilization
in murine hepatocyte cell line. Biochemical Pharmacology, 83(2), 269-278.

Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Examinations:

Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

Details on results:

Non-specific effects
Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).
On HELN cell line 2,4,6-TSP at 10 µM presented low to high non-specific activation of luciferase. A general toxicity was observed for concentrations above 33 µM for TSP.
In the following tests, the maximum concentration tested was set at 33 µM to avoid general cytotoxicity and effects occurring at 10 µM and above for 2,4,6-TSP would be considered as non-specific effects.
On HG5LN cell line, 2,4,6-TSP showed clear non-specific activity at 10 µM and a tendency for non-specific activity at 3 µM. At 33 µM, 2,4,6-TSP showed a general cytotoxic effect assessed by a decrease in luciferase activity relative to control (vehicle DMSO).
In the following tests, the maximum concentration tested was set at 10 µM to avoid general cytotoxicity and effects occurring at 3 and 10 µM would be considered as non-specific effects.

Estrogen receptors activity
Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).
In HELN ERα cells, 2,4,6-TSP did not show any significant agonist activity.
In HELN ERβ cells, 2,4,6-TSP did not show any significant agonist activity.

Thyroid hormone receptors activity
Agonistic activity
Luciferase activity was scaled as percentage of the luciferase activity induced by 100 nm T3. 2,4,6-TSP (range 10 nM -10 µM)did not induce luciferase activity mediated through TRs transactivation.

Antagonistic activity
Antagonistic activity was assessed in the presence of 1 nM T3, a value close to EC50, and expressed as percentage relative to luciferase activity measured in the presence of 100 nM T3 (100 nM T3 provided the maximal transactivation of TRs). An antagonist activity should appear as a downward inflexion of the dose response curve. In HG5LN rTRα cells, 2,4,6-TSP showed a decrease in luciferase activity at 3 µM and 10 µM indicative of a potential antagonistic activity. However this range of concentrations is the range where nonspecific effects started to occur with 2,4,6-TSP.

Pregnane X receptor activity
PXR transactivation was measured relative to the effect of the hPXR agonist 10 µM SR12813 (SR). 2,4,6-TSP presented a specific PXR activity. EC50 estimated for 2,4,6 TSP were 1.71 µM for hPXR, and 1.07 µM for mPXR.

Constitutive androstane receptor activity
hCAR has a high constitutive activity. To show an antagonistic activity, molecules can be directly tested on the HG5LN-hCAR cell line. To detect any putative agonistic activity, constitutive hCAR activity was blocked by the inverse agonist 3 µM PK1195 (IC50=0.71 µM). 2,4,6-TSP did not show specific agonistic or antagonistic activity. The apparent increases in hCAR activity seen at 10 µM are considered to be the result of non-specific effects.
As for its human counterpart, mCAR presents a high constitutive activity, still being stimulated by the agonist TCPOBOP. To enhance the detection of an agonistic activity the inverse agonist T0901317 was used (IC50 1.54 µM). 2,4,6-TSP presented an agonistic activity and was able to increase mCAR activity, with an EC50 value of 4.77 µM. In the presence of the inverse agonist T0901317 at 10 µM, 2,4,6-TSP showed an agonist activity with an EC50 value of 3.13 µM.

Conclusions:

Regarding estrogenic activity of TSP and its impurities/components, only MSP displayed agonist properties on estradiol receptors, however its relative potency was found to be only 10 times less than Bisphenol-A and 20 times less than nonylphenol tested in similar experimental conditions.
TSP presented no specific activities on rat thyroid hormones receptors. TRs are highly conserved across mammals, thus we can extrapolate these results to human TRs.
Regarding xenosensors, TSP was found to induce transactivation of xenosensors mPXR, hPXR and mCAR with EC50 values in the micromolar range (agonist properties similar to cypermethrin).
Overall, these in vitro transactivation studies support the hypothesis that TSP could induce an increase in thyroid hormones turnover in rats due to specificities in rodent endocrinology. Such impact on human thyroid axis is unlikely to occur.

Executive summary:

Many endocrine disruptors exert their harmful effects by interacting with nuclear receptors, which in turn leads to deregulation of gene transcription. Among these nuclear receptors are hormone receptors (glucocorticoid, mineralocorticoid, oestrogen, progestagen and androgens receptors). Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Hormone nuclear receptors, such as thyroid hormone receptors (TRs) and oestrogen receptors (ERs), are well conserved across evolution, especially in mammals. Therefore results obtained with transactivation assays in vitro with a species specific receptor are transposable to other species.

Other nuclear receptors, called xenosensors, are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. These receptors are also involved in hormones turnover and half-life and their activation or repression participate to general endocrine disruption. They are less conserved across evolution and results obtained in transactivation studies are not always transposable to receptors from other species. These molecular variations are the basis of differences observed across species in xenobiotic metabolism.

The aim of the present study was to assess the potential endocrine disrupting properties of tristyrenated phenol (TSP) and its potential impurities or components (mono -MSP- and distyrenated-DSP- phenol).

An in vitro strategy was developed using immortalized luciferase-reporter cell lines expressing various nuclear receptors that could be putative targets for TSP and that could mediate endocrine disruption.

2,4,6-TSP was tested in vitro:

- on the transactivation of human oestrogen receptors (hERα, hERβ) in luciferase reporter cell lines;

- on immortalized cell lines on rat thyroid hormones receptors (rTRα, rTRβ);

- on immortalized cell lines on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination.

Cell lines

All cell lines used in this study derived from the human HeLa immortal cell line. HeLa cell line was derived in 1951 from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows performing comparisons between studies from different laboratories and between studies performed across time (Ballaguer et al., 1999 ).

HELN cell line

HeLa cells were stably transfected with luciferase reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo, giving the HELN cell line, which served as a negative control. HELN cell lines were transfected with estradiol receptor ESR1 (hERα) or ESR2 (hERβ) coding sequence under control of a ubiquitous promoter (Delfosse et al., 2012, Molina-Molina et al., 2013). Hence, ERα or ERβ were expressed and could bind putative estrogenic compounds. When estradiol or xenoestrogen is applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of the ER-α or ER-β. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in the β-globin promoter. Activation of ERE induced the recruitment of transcription regulators and the onset of downstream luciferase expression.

HG5LN cell line

HELA cells were stably transfected with luciferase reporter GAL4 responsive gene: GAL4RE5-βGlob-Luc-SV-Neo, giving the HG5LN cell line which served as a negative control. Chimeric PXR (or TR or CAR) receptors were obtained by fusing the ligand-binding domain (LBD) of PXR (or TR or CAR) with the DNA binding domain (DBD) of GAL4 (Fini et al., 2012, Ménez et al., 2012).

Thus these chimeric receptors upon ligand binding will target the GAL4 Responsive Element (RE) and drive the expression of luciferase gene. This construction has the advantage of being independent from endogenous PXR (TR or CAR) and PRX RE (TR RE or CAR RE). GAL4 is a drosophila gene and is not found in mammalian cells. Transfection of HG5LN with the coding sequence of GAL4-chimeric PXR receptor will lead to the HG5LN-GAL4-PXR sensor cell line. PXR (TR and CAR) belong to the group II of nuclear receptors. They are exclusively located in the nucleus. Upon binding to their ligands, they form heterodimers with XRX receptors and bind to their responsive element. In the presence of a PXR ligand, GAL4 -PXR chimeric receptor will bind to GAL4 RE and induce the recruitment of transcription regulators and the onset of downstream luciferase expression.

Culture conditions

HELN hER-α and HELN hER-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) without phenol red, 1g /L glucose and supplemented with 5% charcoal-dextran treated fetal bovine serum (FCS-DCC) (Molina-Molina et al., 2013), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Delfosse et al., 2012, Molina-Molina et al., 2013). HG5LN GAL4-hPXR, -mPXR, -mCAR, -rTR-α and -rTR-β cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red, 1g/L glucose and supplemented with 5% de-steroidated fetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5mg/mL puromycine and 1mg/mL G418 and kept in a water saturated 5%CO2/95% air atmosphere at 37°C (Fini et al., 2012, Ménez et al., 2012).

Test conditions

All tests were performed in DMEM without phenol red, 1g/L glucose and supplemented with 5% desteroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin.

Cells were plated 24h at 20000 cells/well (200 μL) in 96 wells culture plates (Greiner CellStar) before testing. The day of testing, cells were incubated with the molecules to be tested during 16 hours.

After the incubation, the tested medium was removed and replace with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96 wells plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2 seconds integration time/well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary unit: 100% is given by the reference ligand. Each sample was tested in 4 replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6, 10E-6, 3.16 10E-7, 10E-7, 3.16 10E-8, 10E-8). In case of positive response and thus incomplete curve, successive dilutions were performed until a complete dose response curve was obtained.

These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For hER-α, hER-β, rTR-α and rTR-β, antagonism properties were tested in the presence of 0.1 nM E2 for hER-α, hER-β, and 1nM T3 for rTR-α and rTR-β.

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN and HG5LN). Non-specific effects were quantified relative to vehicle activity (DMSO).

On HELN cell line 2,4,6-TSP at 10 μM presented low to high non-specific activation of luciferase.

A general toxicity was observed for concentrations above 33 μM for TSP and DSPs (2,4-DSP< TSP<2,6-DSP).

In the following tests, the maximum concentration tested was set at 33 μM to avoid general cytotoxicity and effects occurring at 10 μM and above for 2,4,6-TSP would be considered as non-specific effects.

On HG5LN cell line, 2,4,6-TSP showed clear non-specific activity at 10 μM and a tendency for non-specific activity at 3 μM. At 33 μM, 2,4,6-TSP showed a general cytotoxic effect assessed by a decrease in luciferase activity relative to control (vehicle DMSO).

In the following tests, the maximum concentration tested was set at 10 μM to avoid general cytotoxicity and effects occurring at 3 and 10 μM would be considered as non-specific effects.

Estrogen receptors activity

Estrogenic activity revealed by luciferase expression mediated by ER transactivation was quantified in percentage relative to the effect induced by 10 nM 17-β-estradiol (E2).

In HELN ERα cells, 2,4,6-TSP did not show any significant ERα agonist activity.

In HELN ERβ cells, only MSP showed a significant agonist activity starting from 1 μM.

Thyroid hormone receptors activity

Agonistic activity

Luciferase activity was scaled as percentage of the luciferase activity induced by 100 nm T3. None of the three molecules tested (range 10 nM-10 μM) induced luciferase activity mediated through TRs

transactivation.

Antagonistic activity

Antagonistic activity was assessed in the presence of 1 nM T3, a value close to EC50, and expressed as percentage relative to luciferase activity measured in the presence of 100 nM T3 (100 nM T3 provided the maximal transactivation of TRs). An antagonist activity should appear as a downward inflexion of the dose response curve. In HG5LN rTRα cells, 2,4,6-TSP showed a decrease in luciferase activity at 3 μM and 10 μM indicative of a potential antagonistic activity. However this range of concentrations is the range where nonspecific effects started to occur with 2,4,6 -TSP.

Pregnane X receptor activity

PXR transactivation was measured relative to the effect of the hPXR agonist 10 μM SR12813 (SR). The three molecules tested 2,4,6-TSP, 2,4-DSP, 2,6-DSP presented a specific PXR activity: TSP>2,6-DSP>2,4-DSP. EC50 estimated for 2,4,6 TSP, 2,6-DSP and 2,4-DSP were respectively 1.71, 2.3 and 3.67 μM for hPXR, and 1.07, 1.88 and 2.31 μM for mPXR.

Constitutive androstane receptor activity

hCAR has a high constitutive activity. To show an antagonistic activity, molecules can be directly tested on the HG5LN-hCAR cell line. To detect any putative agonistic activity, constitutive hCAR activity was blocked by the inverse agonist 3 μM PK1195 (IC50=0.71 μM). None of the three molecules showed specific agonistic or antagonistic activity. The apparent increases in hCAR activity seen at 10 μM are considered to be the result of non-specific effects.

As for its human counterpart, mCAR presents a high constitutive activity, still being stimulated by the agonist TCPOBOP. To enhance the detection of an agonistic activity the inverse agonist T0901317 was used (IC50 1.54 μM). The three molecules 2,4,6-TSP, 2,4-DSP and 2,6-DSP presented an agonistic activity and were able to increase mCAR activity: 2,4 DSP > 2,6 DSP > 2,4,6 TSP with respective EC50 values: 1.1, 1.44 and 4.77 μM. In the presence of the inverse agonist T0901317 at 10 μM, all three molecules showed an agonist activity: 2,6 DSP = 2,4,6 TSP > 2,4 DSP with respective EC50 values: 2.65, 3.13 and 4.01 μM.

Conclusions

2,4,6-TSP displayed no agonist properties on estradiol receptors.

2,4,6-TSP presented no specific activities on rat thyroid hormones receptors. TRs are highly conserved across mammals, thus we can extrapolate these results to human TRs.

Regarding xenosensors, 2,4,6-TSP was found to induce transactivation of xenosensors mPXR, hPXR and mCAR with EC50 values in the micromolar range (agonist properties similar to cypermethrin).

Overall, these in vitro transactivation studies support the hypothesis that 2,4,6-TSP could induce an increase in thyroid hormones turnover in rats due to specificities in rodent endocrinology. Such impact on human thyroid axis is unlikely to occur.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Case llas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Delfosse, V., Grimaldi, M., Pons, J.-L., Boulahtouf, A., Ie Maire, A., Cavaillès, V., et al.(2012). Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes. Proceedings of the National Academy of Sciences of the USA 109 (37), 14930-14935.

- Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., et al. (2012). Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies.Toxicological Sciences, 125(2), 359-367.

- Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M., & Lespine, A. (2012).Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line. Biochemical Pharmacology, 83(2), 269-278.

- Molina-Molina, J.-M., Amaya, E., Grimaldi, M., Sáenz, J.-M., Real, M., Fernández, M.F., Balaguer, P., Olea, N., 2013. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and Applied Pharmacology 272(1), 127-136.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., & Nicolas, J.-C. (2005). In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry, 340(2), 295-302.

Description of key information

Overall conclusion

In conclusion, based on the available data, there is no relevant concern regarding human ED for any of the MSP, DSP or TSP isomers. Based on in vitro testing data, MSP was the only styrenated phenol which was confirmed to result in slight ER activation, with 4-MSP being much more potent than 2-MSP on both hERalpha and hERbeta. However, when MSP was compared to molecules such as 17beta-estradiol (E2), ethinyl-estradiol (EE2) and Nonylphenols for ER activity, potency was much lower (activities at the micromolar range versus nanomolar range).

An interpretation of all available information related to human health ED properties for the styrenated phenols is available as an attachment to this endpoint summary, and also included in IUCLID Section 13 - Assessment reports.

Furthermore, a comprehensive oral Extended One-Generation Reproduction-Toxicity Study (EOGRTS, OECD 443/408) in rats (Hargitai/CITOX 2018, entry in IUCLID, Section 7.8.1 and 7.5.1) did not provide any evidence of endocrine activity of OAPP, a complex substance, containing the key constituent of concern, namely 4 -(1 -methyl-1-phenylethyl)phenol = p-cumylphenol [CAS no. 599 -64 -4], that is structure-related to 4 -(1-phenylethyl)phenol [4 -MSP, CAS no. 1988 -89 -2]. This study fulfils the criteria of Level 5 within the OECD-ED Conceptual Framework and allowed to evaluate endocrine endpoints of the EATS-modality complex (i.e. oestrogenic, androgenic, thyroidal, and steroidogenic action profiles) along with immunotoxic properties of the test substance.

Based on this analogy approach it is concluded that 4 -MSP and mixtures comprising this key constituent of concern are unlikely to exhibit endocrine activities in rat.

This is further supported by the fact that the target substance, 4 -MSP, unequivocally produced estrogenic effects in zebra fish (see Teigeler/FhI 2018, entry in IUCLID Section 6.6). The corresponding endocrine effect was also observed for the supporting source compound, OAPP, which can be attributed to the presence of the structure-analogous p-cumylphenol (Klix/NOACK 2018, see addendum in IUCLID, Section 6.6).

This suggests that the mono-methylstyrenated phenol is active in fish but not in rat. Therefore, it is assumed that likewise the mono-styrenated phenol (4 -MSP) will fail to exhibit endocrine activity in rats, although active in fish. Hence, it is further concluded that the complex target substance, phenol, styrenated, containing 4 -MSP, is unlikely to show endocrine properties in the rat (see 7.9.4 Attached Document: overview table on structure and endocrine effects relationship).

A detailed justification of the analogy approach, OAPP vs. Phenol, styrenated / p-cumylphenol vs. 4 -MSP, is provided in the respective sections, in IUCLID, 7.8.1 and 7.5.1. Endocrine effects data on fish are found in IUCLID under section 6.6.