9,9-Bis(methoxymethyl)-9H-fluorene

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

single dose application, 48 h incubation of yeast cells

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

comparable to OECD 455

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 455

Version / remarks:

the YES/YAS assay works with genetically modified yeast cells (human estrogen and androgen receptors hERa and hAR, reporter gene lac-Z for ß-galactosidase)

Deviations:

not applicable

Principles of method if other than guideline:

The YES/YAS assay uses genetically modified yeast cells, that can interact with the human estrogen and androgen receptors hERa and hAR. The reporter gene lac-Z, which encodes the enzyme ß-galactosidase is also integrated in the yeast cells.
If a test item interacts with the estrogen or androgen receptors, the reporter gene will be expressed and ß-galactosidase will be secreted into the medium. The medium contains an indicator (CPRG), which is converted by ß-galactosidase into a red product. This product can be measured photometrically.
The measured OD (at 570 nm) values correlate directly with the amount of produced ß-galactosidase, which is an indication of the test item's activity towards the hERa or hAR receptors.

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Kaiser-Friedrich-Str. 7, D-55116 Mainz, Germany

Type of method:

in vitro

Test material

Test material information

Constituent 1

Reference substance name:

9,9-Bis(methoxymethyl)-9H-fluorene

EC Number:

682-678-3

Cas Number:

182121-12-6

Molecular formula:

C17H18O2

IUPAC Name:

9,9-Bis(methoxymethyl)-9H-fluorene

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

9,9-Bis(methoxymethyl)-9H-fluorenone, CAS No. 682-678-3, monoconstituent substance, homogeneous, solid, stable at room temperature
batch no. Mel-Sab-01, expiry date Dec. 2018, purity: >99%

Test animals

Species:

other: yeast (Saccharomyces cerevisiae), genetically modified

Strain:

other: yeast (Saccharomyces cerevisiae), genetically modified

Remarks:

hERa and hAR receptor, expression plasmid carrying the reporter gene lacZ

Sex:

not specified

Details on test animals or test system and environmental conditions:

The YES-YAS Assay Kit including Saccharomyces cerevisiae was obtained from Xenometrix AG, Gewerbestraße 25, CH-4123 Allschwil, Switzerland
batch No:: N05-12477
4 days before the start of the experiment, the yeast cultures were prepared in 2 different culture flasks (for YES and YAS assays respectively) and incubated at 32 +/- 1°C for 71h (YAS culture) or 95.5 h (YES culture) until the cultures were clearly turbid. Then the optical density at 690 nm for the growth of the yeast cells was measured with a microplate reader. The cultures were diluted appropriately and used for the experiment.

Administration / exposure

Route of administration:

other: dropped into culture cultureflasks

Vehicle:

DMSO

Details on exposure:

After checking the yeast cultures for growth with a microplate reader by measuring the optical density, 7 dilutions of positive controls and 8 dilutions of test item were pipetted into a 96-well dilution plate.
Next, 4 assay plates were labelled (YES Agonist/ YAS Agonist/ YES Antagonist/ YAS Antagonist) and filled with the corresponding assay medium. Then each positive control dilution and test item dilution was pipetted from the dilution plate to an assay plate in duplicate. After addition of the yeast cells the plates were incubated at 32 +/- 1°C in a humidity container with agitation. Colour change of the medium in the positive control wells indicated growth after 46 h.
Then, the well plates were measured with a microplate reader at 690 nm for cell growth and at 570 nm for colour development.
Evaluation was done with Microsoft Excel.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

microplate reader at 690 nm for cell growth and at 570 nm for colour development of positive controls and test item.

Duration of treatment / exposure:

46 h (indication by colour change of the positive controls)

Frequency of treatment:

single dose application

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mol/L

Remarks:

8 concentrations with half-log concentrations, up to 0.0001 mol/L

Doses / concentrations 2

Dose / conc.:

0 other: mol/L

Doses / concentrations 3

Dose / conc.:

0 other: mol/L

Doses / concentrations 4

Dose / conc.:

0 other: mol/L

Doses / concentrations 5

Dose / conc.:

0 other: mol/L

Doses / concentrations 6

Dose / conc.:

0 other: mol/L

Doses / concentrations 7

Dose / conc.:

0 other: mol/L

Doses / concentrations 8

Dose / conc.:

0 other: mol/L

Details on study design:

8 dilutions of positive controls or test item were examined in duplicate.
YES or YAS agonistic activity was detected directly by ß-galctosidase activity. Antagonistic activities were detected by inhibition of ß-galactosidase activities in the presence of estrogenic or androgenic substances.
The solvent DMSO served as negative control. Cytotoxicity was checked by measurement of the cell density.

Examinations

Examinations:

Colour change by ß-galactosidasse activity
The ß-galactosidase activity correlates directly with the activating (agonist) or inhibiting (antagonist) activity of the test item.

Positive control:

YES Agonist assay: 17ß-estradiol (E2)
YES Antagonist assay: 4-hydroxytamoxifen (HT)
YAS Agonist assay: 5a-dihyrotestosterone (DHT)
YAS Antagonist assay: Flutamide (FL)

Results and discussion

Details on results:

The experiment was valid. 8 dilutions of the test item in DMSO and controls (solvent control and positive controls) were exposed to the modified yeast cells in duplicate. After incubation positive and solvent (negative) controls showed the expected results. No cytotoxicity was observed (controlled by measurement of the cell density). The test item showed no positive result in any assay. The values of ß-galactosidase activity and induction ratio of all test item dilutions lay in the same range as the values of the respective controls. No dose-response correlation was visible.
Therefore the test item is considered to have no estrogenic and androgenic agonistic and antagonistic activity under the conditions of this experiment.

Applicant's summary and conclusion

Conclusions:

The test item is considered to show no estrogenic or androgenic agonistic or antagonistic activities in the YES/YAS assay.