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additional toxicological information
Remarks:
Estrogenic Agonist and Antagonist Activity (in vitro)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Dec 2016 - 31 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Type of study / information:
Evaluation of estrogenic Agonist and Antagonist Activity of using the Stably Transfected Human Estrogen Receptor-α Transactivation Assay (hERα-HeLa-9903 Cell Line)
Test guideline
Qualifier:
according to
Guideline:
other: OECD No. 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists
Version / remarks:
Adopted July 29 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Results and discussion

Any other information on results incl. tables

Determination of cytotoxicity using the MTT test

In the first experiment it was observed that the test item was not cytotoxic up to a concentration of 10 µM. However, at 100 µM cytotoxic effects (22% decrease in viability) were observed. Based on this result it was decided to test the test item up to 10 µM in (ant)agonist exposure experiment 1.

Based on the outcome of ER (ant)agonist experiment 1 it was decided to better characterize the potential estrogenic (ant)agonistic effects in the range between 10 and 100 µM. Therefore, the MTT test was repeated with inclusion of 20, 30, 40 and 50 µM as additional test item concentrations. At 100 µM, again cytotoxicity was observed (32%). No cytotoxicity was observed at all other test concentrations. However, there was again a slight decrease in the number of cells observed in the wells exposed to 50 and 100 µM test item. Therefore, it was decided to use 30 µM as highest test concentration in ER (ant)agonist experiment 2 and 3.

ER agonist assay

The results of the ER agonist assay are summarised in Table 1 below. The mean luciferase activity of the positive control (1 nM E2), included in each experiment was at least 4-fold higher than the vehicle control in all experiments (18.31, 18.04 and 13.44-fold for the QC-plates, and 18.70, 17.39 and 9.50-fold for the test item plates). The fold induction of E2 corresponding to the PC10value was above the fold induction of the vehicle control +2SD (standard deviations) in all experiments. Therefore, all results obtained were accepted.

The results of E2 and corticosterone were within the acceptability criteria. The curve fit value log log PC10 for 17α-estradiol was in experiment 2 just outside the acceptability criteria mentioned in the guideline, but as the results of the three independent experiments with 17α-estradiol were comparable, the results were accepted. The curve fit values log PC50, log PC10 for 17α‑methyltestosterone were outside theacceptability criteria mentioned in the guideline, but were within our laboratory historical control data range and thus accepted. The reference items were correctly classified as positive (E2, 17α-estradiol and 17α-methyl-testosterone) or negative (corticosterone).

The RPCmax values obtained for the test item were below 10% in all three independent experiments. As such, 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO was considered to be negative in the ER agonist assay when tested up to a concentration of 30 µM.

Table 1: Log PC50, Log PC10, Log EC50, Hill slope and RPCmax values obtained for reference items and test item in the ER agonist assay (three independent experiments)

Name

Experiment

number

log
PC50

log PC10

log EC50

Hill slope

RPCmax(%)

Judgement

17β-Estradiol (E2)

1

-10.76

-11.90

-10.88

1.46

99.3

Positive

2

-10.54

-11.68

-10.51

1.34

112

Positive

3

-10.43

-11.46

-10.41

1.07

104

Positive

17α-Estradiol

1

-8.32

-10.65

-8.41

1.30

101

Positive

2

-8.49

-9.24

-8.50

1.71

108

Positive

3

-8.45

-9.32

-8.50

1.44

107

Positive

17α-Methyl-testosterone

1

-

-5.80

-

-

33.6

Positive

2

-

-5.43

-

-

16.6

Positive

3

-

-5.67

-

-

24.0

Positive

Corticosterone

1

-

-

-

-

1.5

Negative

2

-

-

-

-

1.0

Negative

3

-

-

-

-

-0.2

Negative

4,4'-Isopropylidene-diphenol, propoxylated, Grade 4PO

1

-

-

-

-

7.1

Negative

2

-

-

-

-

5.5

Negative

3

-

-

-

-

3.8

Negative

PC50=  concentration inducing 50% of the maximum level of the positive control; PC10= concentration inducing 10% of the maximum level of the positive control; EC50= concentration of agonist that induces a response halfway between the baseline (bottom) and maximum (top) response; RPCmax= maximum level of response induced by the compound, expressed as percentage of the response induced by 1 nM E2

ER antagonist assay

The results of the ER antagonist assay are summarised in Table 2 below. The mean luciferase activity of the spike-in control (AGref) included in each experiment was at least 4-fold higher than the vehicle control in experiments 1 and 2 (4.9-, 5.3-fold in experiment 1 and 4.2- and 4.8-fold and in experiment 2). In experiment 3, the mean luciferase activity of the AGref control on the reference item plate and test item plate was 3.3-fold and 3.6-fold the mean vehicle control. This was outside the ≥ 4-fold acceptance criteria and therefore the results of ER antagonist experiment 3 were rejected.

In ER antagonist assay experiment 1 and 2 all acceptability criteria were met, or were in accordance with laboratory historical data. The reference items were correctly classified as positive (tamoxifen and OHT) or negative (flutamide). Therefore, these experiments were considered to be valid.

For 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO no IC30 could be determined. This was reproducible in two independent experiments. As such, 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO is considered to be negative in the ER antagonist assay when tested up to a concentration of 10 µM (experiment 1) and 30 µM (experiment 2).

Table 2: Log IC50 and log IC30 values of the reference items tamoxifen, flutamide and OHT obtained in the ER antagonist assay

Name

Experiment number

Log IC50

Log IC30

Judgement

Tamoxifen

1

-5.99

-6.59

Positive

2

-5.91

-6.79

Positive

OHT

1 (reference item plate)

-7.80

-8.45

Positive

1 (test item plate)

-8.22

-8.58

Positive

2 (reference item plate)

-

-7.92

Positive

2 (test item plate)

-8.18

-8.73

Positive

Flutamide

1

-

-

Negative

2

-

-

Negative

4,4'-Isopropylidene-diphenol, propoxylated, Grade 4PO

1

-

-

Negative

2

-

-

Negative

IC50= half maximum effective concentration of an inhibitory test item; IC30= concentration inhibiting 30% of the maximum level of the positive control.

Applicant's summary and conclusion

Conclusions:
In the ER agonist assay all acceptability criteria were met, or were in accordance with laboratory historical data. In addition, all four reference items were correctly classified as positive or negative. As such it was concluded that all ER agonist assay data were valid. Since the RPCmax values were below 10% in all three independent experiments, 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO is considered to be negative in the ER agonist assay when tested up to a concentration of 30 µM.

In ER antagonist assay experiment 1 and 2 all acceptability criteria were met, or were in accordance with laboratory historical data. The results of ER antagonist experiment 3 were rejected since the response of the spike-in control (AGref) was outside the acceptability criteria. In experiment 1 and 2, the reference items were correctly classified as positive or negative. Therefore, these experiments were considered to be valid. Since no IC30 could be determined, 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO was considered to be negative in the ER antagonist assay when tested up to a concentration of 10 µM (experiment 1) and 30 µM (experiment 2).

It can be concluded that 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO does not show any estrogenic or anti-estrogenic activity when tested in stably transfected human Estrogen-α Transactivation Assay.
Executive summary:

The ability of 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO to establish signal activation or blocking of the estrogen receptor was investigated in a GLP compliant study in accordance with OECD guideline Test No. 455. The study used the human hERα-HeLa-9903 cell line, which is stably transfected with the human Estrogen Receptor α coupled to a firefly luciferase receptor gene construct. For both the ER agonist and ER antagonist assay three independent assays were performed. Within each ER agonist experiment, the test item was tested at seven concentrations (each concentration in triplicate) ranging from 10 pM to 10 µM (experiment 1) or 100 pM to 30 µM (experiments 2 and 3). Positive controls 17ß-estradiol (E2), 17α-estradiol, 17α-methyltestosterone and negative control corticosterone were tested in the ER agonist assay. Within each ER antagonist experiment, the test item was tested at six concentrations (each concentration in triplicate) ranging from 100 pM to 10 µM (experiment 1) or 1 nM to 30 µM (experiments 2 and 3). Taxoxifen and OHT were employed as positive controls and Flutamide as a negative control. All assays also included a vehicle control (DMSO). Two MTT assays were also carried out to determine the cytotoxity of the test substance. It can be concluded that 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO does not show any estrogenic or anti-estrogenic activity when tested in stably transfected human Estrogen-α Transactivation Assay.

Three out of the three ER agonist assays met acceptability criteria. Since the RPCmax values were below 10% in all three independent experiments, 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO is considered to be negative in the ER agonist assay when tested up to a concentration of 30 µM. Only two of the three antagonist assays met the acceptability criteria. The results of the third ER antagonist assay were therefore disregarded. Since no IC30 could be determined, 4,4'-Isopropylidenediphenol, propoxylated, Grade 4PO was considered to be negative in the ER antagonist assay when tested up to a concentration of 10 µM (experiment 1) and 30 µM (experiment 2).