3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

specific investigations: other studies

Remarks:

Evaluation of ER agonist or antagonist activity.

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 September 2017 - 11 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists

Deviations:

no

GLP compliance:

not specified

Type of method:

in vitro

Specific details on test material used for the study:

Storage conditions: Room temperature.

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Agonist (AG) positive control:
1) 17β-Estradiol (Abbreviation: E2)
2) 17α-Estradiol (Abbreviation: α-E2)
3) 17α-Methyltestosterone (Abbreviation: MT)

- Agonist (AG) negative control:
1) Corticosterone (Abbreviation: Cor)

- Antagonist (ATG) positive control:
1) Tamoxifen (Abbreviation: Tam)
2) 4-Hydroxytamoxifen (Abbreviation: OHT)

- Antagonist (ATG) negative control:
1) Flutamide (Abbreviation: Flu)

- Cytotoxicity positive control:
1) Digitonin (Abbreviation: Dig)

- Vehicle control
1) Dimethyl sulfoxide (Abbreviation: DMSO)

CELL LINE
- hERα-HeLa9903

CULTURE MEDIUM
- Eagle’s Minimal Essential Medium without phenol red (Nissui) was supplemented with 10% heat-inactivated Charcoal/Dextran treated FBS (Hyclone) and sterilized by filtration.

Details on study design:

STUDY CONDITIONS
- Reagents
1) Luciferase activity: Steady-Glo Luciferase assay system (Promega)
2) Cytotoxicity: Cell Counting Kit-8 (Dojindo)
- Instrument: Microplate reader (PerkinElmer, ARVOTM X4)

TEST METHODS
- Preparation of DMSO solutions of test material and control substances:
1) Test material: 10 mM solution was selected as the maximum concentration of stock solution because 1 M and 100 mM solutions were precipitated in culture medium. Then 10 mM TG-MD solution was serially diluted with DMSO at a ratio of 1:10, and seven doses for ER AG assay or six doses for ER ATG assay were prepared.
2) Control substances: Each of the chemicals were dissolved in DMSO and 10 mM stock solutions (for E2, α-E2, MT, Tam, OHT and Flu) or 100 mM stock solutions (for Cor and Dig) were prepared. Then each stock solution was serially diluted with DMSO at a ratio of 1:10. For ER AG assay, E2, α-E2, MT and Cor solutions were prepared at seven doses. For ER ATG assay, Tam and Flu solutions were prepared at six doses. OHT solution was prepared at 1 mM.

ER AG ASSAY / LUCIFERASE ASSAY
- HeLa9903 cells were added to each well of 96 well white plate at a density of 10000 cells/100 μL/well.
- The plates were incubated in a 5 % CO2 incubator at 37 °C for 3 hours.
- The chemicals were diluted (test material and control substances) in culture medium at a ratio of 3:1000 and 50 μL of the diluted chemicals was added to the plate.
- The plate was incubated in a 5 % CO2 incubator at 37 °C for 20-24 hours.
- All the medium was removed from the plate and 50 μL of Steady-Glo Luciferase assay system reagent diluted with phosphate-buffered saline (PBS) was added.
- The plate was left at room temperature for 10 minutes and the luminescence measured by microplate reader.

ER ATG ASSAY
1) Luciferase assay:
- HeLa9903 cells were added to each well of 96 well white plate at a density of 10000 cells/100 μL/well.
- The plate was incubated in a 5 % CO2 incubator at 37 °C for 3 hours.
- The chemicals were diluted in 75 pM E2 supplemented culture medium at a ratio of 3:1000 and 50 μL of the diluted chemicals were added to the plate.
- The plate was incubated in a 5 % CO2 incubator at 37 °C for 20-24 hours.
- All the medium was removed from the plate and 50 μL of Steady-Glo Luciferase assay system reagent diluted with PBS was added.
- The plate was left at room temperature for 10 minutes and the luminescence measured by microplate reader.

2) Cytotoxicity assay
- HeLa9903 cells were added to each well of 96 well clear plate at a density of 10000 cells/100 μL/well.
- The chemicals were added to the plate and incubated in the same way as in the luciferase assay.
- All the medium was removed from the plate and 150 μL of Cell Counting Kit-8 reagent diluted with culture medium was added.
- The plate was incubated in a 5 % CO2 incubator at 37 °C for 90 minutes and the absorbance at 450 nm was measured by microplate reader.

DATA ANALYSIS
- The mean value of relative luminescence unit (RLU) for vehicle control (VC) was subtracted from each well RLU value. Then relative transcriptional activity (RTA) was calculated using the following formulae:

RTA (%) for AG assay = Experimental RLU value / RLU value of PC (1 nM E2) x 100
RTA (%) for ATG assay = Experimental RLU value / RLU value of SC (25 pM E2) x 100

- For the AG assay, if the Hill’s logistic equation is applicable to the concentration response data, the half maximal effective concentration (EC50) was calculated in a statistical software (GraphPad PRISM GraphPad software Inc.).
- The concentration of chemical at which the measured activity is 10 % or 50 % of the maximum activity induced by the PC (referred to as PC10 or PC50) was calculated by interpolating between 2 points on the X-Y coordinate, one immediately above and one immediately below a PC10 or PC50 value.
- If the PC10 failed to calculate (the maximum RTA induced by the test material was less than 10 %) in two of two experiments, the test material was evaluated as negative in ER AG assay.

- For the ATG assay, the concentration of chemical at which the measured activity is 70 % or 50 % of the maximum activity induced by the SC (referred to as IC30 or IC50) was calculated by interpolating between 2 points on the X-Y coordinate, one immediately above and one immediately below a IC30 or IC50 value.
- If the IC30 failed to calculate (the minimum RTA induced by the test material was greater than 70 %) in two of two experiments, the test material was evaluated as negative in ER ATG assay.
- For the cytotoxicity assay, the mean value of absorbance (Abs) for cytotoxicity control (Cx) was subtracted from each well Abs value. Then cell viability was calculated using the following formulae:

Cell viability (%) = (Abs chemical – Abs cx) / (Abs SC – Abs cx) x 100

Details on results:

ER AG assay (Table 1, Table 2, Table 3 and Table 4):
- The values of logPC50, logPC10, logEC50 and Hill slope of the test material were not calculated in all experiments and the test material was evaluated as negative in ER AG assay.
- The values of logPC50, logPC10, logEC50 and Hill slope of control substances (E2, α-E2, Cor and MT) were summarised in Table 3 and Table 4.
- The values of fold induction of PC (1 nM E2) were 19.0 (plate 1) and 18.3 (plate 2) in the first experiment and 18.4 (plate 1) and 19.6 (plate 2) in the second experiment.
- The values of fold induction corresponding to the PC10 value of the concurrent PC (1 nM E2) were 2.8 (plate 1) and 2.7 (plate 2) in the first experiment and 2.7 (plate 1) and 2.9 (plate 2) in the second experiment.
- Therefore, all acceptance criteria was passed.

ER ATG assay (Table 5, Table 6, Table 7, Table 8, Table 9 and Table 10)
- The values of logIC50 and logIC30 of the test material were not calculated in all experiments and the test material was evaluated as negative in ER ATG assay.
- The values of fold induction of SC (25 pM E2) were 8.2 in the first experiment and 7.7 in the second experiment.
- The RTA of AG-PC (1 nM E2), ATG-PC (1 μM OHT) and Cx (100 μM Dig) were 236.04 %, 29.21 % and -12.23 % in the first experiment and 235.68 %, 25.29 % and -13.16 % in the second experiment.
- Therefore, all acceptance criteria was passed.
- Cytotoxicity was not shown in the test material and all control substances because cell viability was above 80 %.

Table 1. RTA (%) in ER AG assay (First experiment)

E2
Log M	Mean	SD
-8	98.0	2.9
-9	96.7	4.8
-10	81.2	2.4
-11	30.3	5.9
-12	6.6	2.9
-13	1.9	0.9
-14	0.6	1.6

α-E2
Log M	Mean	SD
-6	91.3	1.8
-7	87.6	7.2
-8	77.6	6.4
-9	36.5	2.7
-10	5.3	1.8
-11	1.9	1.1
-12	-0.2	1.0

Cor
Log M	Mean	SD
-4	-3.3	0.4
-5	-1.5	0.3
-6	-1.3	0.5
-7	-0.4	0.2
-8	0.1	1.5
-9	0.0	0.2
-10	-0.3	1.1

MT
Log M	Mean	SD
-5	67.0	1.7
-6	13.3	0.8
-7	6.5	2.1
-8	2.1	2.1
-9	0.3	1.7
-10	0.4	1.4
-11	0.4	0.7

Test material
Log M	Mean	SD
-5	0.1	0.3
-6	0.0	0.1
-7	0.1	0.5
-8	0.1	0.6
-9	0.5	0.6
-10	0.7	0.8
-11	0.4	1.2

 

Table 2. RTA (%) in ER AG assay (Second experiment)

E2
Log M	Mean	SD
-8	103.1	4.2
-9	100.1	1.0
-10	85.6	0.7
-11	35.9	5.1
-12	8.0	2.5
-13	2.3	0.7
-14	0.1	1.2

α-E2
Log M	Mean	SD
-6	99.5	0.6
-7	98.0	6.3
-8	87.7	4.0
-9	38.5	2.8
-10	6.8	1.1
-11	3.0	0.9
-12	0.2	0.4

Cor
Log M	Mean	SD
-4	-1.4	0.3
-5	0.0	0.1
-6	-0.5	0.9
-7	0.4	0.4
-8	0.5	0.7
-9	0.3	0.2
-10	0.6	1.0

MT
Log M	Mean	SD
-5	72.8	1.5
-6	17.0	1.1
-7	7.4	1.4
-8	2.9	1.1
-9	0.9	0.7
-10	1.0	1.0
-11	0.6	0.3

Test material
Log M	Mean	SD
-5	0.3	0.4
-6	0.4	0.3
-7	0.2	0.3
-8	0.4	0.5
-9	0.8	0.1
-10	0.4	0.5
-11	0.3	0.1

 

Table 3. The values of logPC50, logPC10, logEC50 and Hill slope in ER AG assay (First experiment)

	LogPC50	LogPC10	LogEC50	Hill slope
E2	-10.6 (-11.4 ~ -10.1)	-11.9 (<11)	-10.7 (-11.3 ~ -10.1)	1.0 (0.7 ~ 1.5)
α-E2	-8.7 (-9.6 ~ -8.1)	-9.9 (-10.7 ~ -9.3)	-8.8 (-9.6 ~ -8.4)	1.0 (0.9 ~ 2.0)
Cor	NC (－)	NC (－)	NC (－)	NC (－)
MT	-5.3 (-6.0 ~ -5.1)	-6.5 (-8.0 ~ -6.2)	NC (－)	NC (－)
Test material	NC	NC	NC	NC

*( ) shows the acceptance criteria in TG455.

*NC: Not Calculated

 

Table 4. The values of logPC50, logPC10, logEC50 and Hill slope in ER AG assay (Second experiment)

	LogPC50	LogPC10	LogEC50	Hill slope
E2	-10.7 (-11.4 ~ -10.1)	-11.9 (<11)	-10.7 (-11.3 ~ -10.1)	0.9 (0.7 ~ 1.5)
α-E2	-8.8 (-9.6 ~ -8.1)	-9.9 (-10.7 ~ -9.3)	-8.8 (-9.6 ~ -8.4)	1.0 (0.9 ~ 2.0)
Cor	NC (－)	NC (－)	NC (－)	NC (－)
MT	-5.4 (-6.0 ~ -5.1)	-6.7 (-8.0 ~ -6.2)	NC (－)	NC (－)
Test material	NC	NC	NC	NC

*( ) shows the acceptance criteria in TG455.

*NC: Not Calculated

 

Table 5. RTA (%) in ER ATG assay (First experiment)

Tam
Log M	Mean	SD
-5	21.6	6.4
-6	47.3	1.5
-7	82.2	7.5
-8	115.1	17.3
-9	105.6	9.5
-10	106.5	8.2

Flu
Log M	Mean	SD
-5	90.5	13.6
-6	104.5	9.2
-7	107.9	6.9
-8	105.6	3.8
-9	106.3	6.2
-10	106.2	3.9

Test material
Log M	Mean	SD
-5	89.2	5.9
-6	104.7	15.4
-7	100.7	13.0
-8	102.3	7.7
-9	95.8	14.5
-10	102.4	4.3

 

Table 6. RTA (%) in ER ATG assay (Second experiment)

Tam
Log M	Mean	SD
-5	21.1	3.4
-6	44.3	2.4
-7	80.0	5.6
-8	105.1	4.7
-9	104.2	7.8
-10	104.4	6.1

Flu
Log M	Mean	SD
-5	90.1	6.2
-6	98.6	4.3
-7	103.2	4.2
-8	103.5	1.7
-9	103.0	2.9
-10	104.1	2.7

Test material
Log M	Mean	SD
-5	89.7	3.6
-6	95.1	3.4
-7	99.6	9.4
-8	98.3	5.4
-9	103.9	8.4
-10	97.9	5.0

 

Table 7. Cell viability (%) in ER ATG assay (First experiment)

Tam
Log M	Mean	SD
-5	85.4	1.9
-6	90.2	3.1
-7	95.8	4.7
-8	96.8	4.0
-9	98.1	7.3
-10	97.0	2.5

Flu
Log M	Mean	SD
-5	93.5	1.1
-6	90.6	1.3
-7	93.1	4.6
-8	95.2	1.4
-9	98.9	0.5
-10	98.6	3.0

Test material
Log M	Mean	SD
-5	96.3	4.1
-6	101.9	14.9
-7	101.8	7.6
-8	98.9	4.3
-9	97.1	3.8
-10	97.4	3.1

 

Table 8. Cell viability (%) in ER ATG assay (Second experiment)

Tam
Log M	Mean	SD
-5	92.2	2.1
-6	95.5	2.0
-7	95.7	4.2
-8	99.4	0.3
-9	98.2	2.9
-10	98.9	1.0

Flu
Log M	Mean	SD
-5	82.5	4.9
-6	93.7	1.2
-7	95.8	1.0
-8	99.4	0.7
-9	99.4	0.5
-10	98.6	3.1

Test material
Log M	Mean	SD
-5	93.5	0.6
-6	98.0	3.7
-7	98.9	4.0
-8	97.6	2.3
-9	99.3	0.2
-10	98.4	0.9

 

Table 9. The values of logIC50 and logIC30 in ER ATG assay (First experiment)

	LogIC50	LogIC30
Tam	-6.08 (-7.596 ~ -5.942)	-6.65 (－)
Flu	NC (－)	NC (－)
Test material	NC	NC

*( ) shows the acceptance criteria in TG455.

*NC: Not Calculated

 

Table 10. The values of logIC50 and logIC30 in ER ATG assay (Second experiment)

	LogIC50	LogIC30
Tam	-6.16 (-7.596 ~ -5.942)	-6.72 (－)
Flu	NC (－)	NC (－)
Test material	NC	NC

*( ) shows the acceptance criteria in TG455.

*NC: Not Calculated

Conclusions:

Under the conditions of this study the test material was evaluated as negative in ER AG assay and in ER ATG assay.

Executive summary:

Estrogen receptor (ER) agonist (AG) or antagonist (ATG) activity of the test material was evaluated in stably transfected transactivation in vitro assays using hERα-HeLa9903 cell line according to the standardised guidelines OECD 455. 

The test material was tested twice at the concentration range of 10^-5～10^-11 M because 10^-3 and 10^-4 M test material solutions were precipitated in cell culture medium.

As the concentration of the test material at which the measured activity in an ER AG assay is 10 % of the maximum activity induced by the 1nM E2 (PC10) and 30 % maximal effective concentration of an inhibitory test material in an ER ATG assay, (IC30) were not calculated.

Under the conditions of this study the test material was evaluated as negative in ER AG assay and in ER ATG assay.

Description of key information

Evaluation of ER agonist or antagonist activity (Maeda, 2017)

The test material was evaluated as negative in ER AG assay and in ER ATG assay.

Additional information

Evaluation of ER agonist or antagonist activity (Maeda, 2017)

Estrogen receptor (ER) agonist (AG) or antagonist (ATG) activity of the test material was evaluated in stably transfected transactivation in vitro assays using hERα-HeLa9903 cell line according to the standardised guidelines OECD 455. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested twice at the concentration range of 10^-5～10^-11 M because 10^-3 and 10^-4 M test material solutions were precipitated in cell culture medium.

As the concentration of the test material at which the measured activity in an ER AG assay is 10 % of the maximum activity induced by the 1nM E2 (PC10) and 30 % maximal effective concentration of an inhibitory test material in an ER ATG assay, (IC30) were not calculated.

Under the conditions of this study the test material was evaluated as negative in ER AG assay and in ER ATG assay.