4,4'-methylenedianiline

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The test was carried out based on the description of P. Sohoni and J.P. Sumpter [Sohoni, P.; Sumpter, J.P.: Several environmental oestrogens are also anti-androgens. J. Endocrinol., Vol. 158, No. 3, pp. 327-339, 1998]. The test has been validated at the test facility [Kolle S.N., Kamp H.G., Huener H.-A., Knickel J., Verlohner A., Woitkowiak C., Landsiedel R., v. Ravenzwaay B.: In house validation of recombinant yeast estrogen and androgen receptor agonist and antagonist screening assays. Toxicol In Vitro, Vol. 24, No. 7, pp 2030-2040, 2010].

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: androgenic and / or antiandrogenic potential of the test substance

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenedianiline

EC Number:

202-974-4

EC Name:

4,4'-methylenedianiline

Cas Number:

101-77-9

Molecular formula:

C13H14N2

IUPAC Name:

4,4'-methylenedianiline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1435367
- Purity: 98 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

OTHER SPECIFICS: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.

Test animals

Species:

other: hAR yeast strain

Strain:

other: not applicable

Administration / exposure

Route of administration:

other: not applicable

Vehicle:

DMSO

Remarks:

(concentration in culture medium: 1%)

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): DMSO was used as vehicle, which had been demonstrated to be suitable in the yeast androgen screen up to 1% (v/v) and for which historical control data are available.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

48 h (+/- 4 h)

Frequency of treatment:

once

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

Doses / concentrations 2

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

Doses / concentrations 3

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

Doses / concentrations 4

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

Doses / concentrations 5

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

Doses / concentrations 6

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

Doses / concentrations 7

Dose / conc.:

0 other: mol/L

Remarks:

nominal conc.

No. of animals per sex per dose:

not applicable

Control animals:

other: vehicle control

Details on study design:

Yeast cells (Saccharomyces cerevisiae) have been stably transformed with a gene encoding the human androgen receptor (hAR), which is constitutively expressed. Additionally, these cells are stably transformed with a reporter gene plasmid, containing an androgen response element and the LacZ gene, which encodes the reporter enzyme β-galactosidase (The hAR yeast strain was obtained from “Technische Universität Dresden”, Prof. Dr. G. Vollmer on 11 Feb 2010.). The hAR yeast strain are checked for androgenic and antiandrogenic activity at regular intervals. Additionally the responsiveness of the yeast strain was verified in each experiment using concurrent positive controls.

A deep-frozen (-80°C) yeast stock culture was thawed at room temperature, inoculated in growth medium and incubated for preculture (24 - 72 h) and growth medium was exchanged after 72 h before use. Of the preculture, optical density (OD) was determined at 690 nm. For preparation of the test culture, 0.50 mL of the preculture with an OD of 1.0 was transferred into 50 mL fresh culture medium including 0.5 mL chromogenic substrate CPRG (chlorophenol red-β-D-galactopyranoside). The study was carried out in 96-well microtiter plates in which 2 μL of different test substance solutions had been pipetted. 200 μL of the test culture was added to each well. The plates were sealed with breathable tape and incubated. Two independent experiments (with 4 replicates per concentration in each experiment) were conducted.

Examinations

Examinations:

After 48 h (+/- 4 h) incubation, absorbance of the plates was measured at 570 nm (color development, androgen receptor dependent enzyme expression) and 690 nm (turbidity due to growth of the yeast). Evaluation was performed by calculating the difference of the measured ODs at the two wavelengths (absorption at 570 nm - absorption at 690 nm).
Cytotoxicity of the test substance is generally indicated by a decrease in the yeast growth (measurement of the turbidity at 690 nm). For the evaluation of antiandrogenic activity only nontoxic test substance concentrations are taken into consideration.
Possible test substance precipitation was examined microscopically in the test plates.

Acceptance criteria:
Generally, the experiment is considered valid, if the following criteria are met:
1.) The positive controls induced an agonistic / antagonistic effect within the range of the historical control data.
2.) The concentration 5*10E-09 mol/L 5α-dihydrotestosterone achieved at least 40% of the maximum effected androgen receptor dependent enzyme expression of the positive control (color development) based on the experiment.
3.) The vehicle control did not show color development at 570 nm.

Assessment criteria:
Generally, cytotoxicity (decrease of the yeast growth) is considered for data interpretation especially in the case of antiandrogenic activity. A test substance is generally considered non-androgenic in this assay, if:
1.) Androgen receptor dependent enzyme expression was within the historical negative control range under all experimental conditions in two experiments carried out independently.
The test substance is considered as androgenic in this assay, if:
1.) A concentration-dependent and reproducible increase of the androgen receptor dependent enzyme expression (color development) by at least 20% compared to the vehicle control was observed.
2.) If a concentration-dependent and reproducible increase of the androgen receptor dependent enzyme expression (color development) by at least 10% and less than 20% compared to the vehicle control was observed, the test substance is considered to be slightly androgenic.
The test substance is considered as antiandrogenic in this assay, if:
1.) A concentration-dependent and reproducible inhibition of the androgenic effect compared to 5*10E-09 mol/L 5α-dihydrotestosterone (partly or total suppression of expected color development, without signs for cytotoxicity) by at least 20% was observed compared to 5*10E-09 mol/L 5α-dihydrotestosterone alone.
2.) If a concentration-dependent and reproducible inhibition of the androgenic effect compared to 5*10E-09 mol/L 5α-dihydrotestosterone (partly or total suppression of expected color development, without signs for cytotoxicity) by at least 10% but less than 20% compared to 5*10E-09 mol/L 5α-dihydrotestosterone alone was observed, the test substance is considered to be slightly antiandrogenic.

Positive control:

Androgenic control: 5α-dihydrotestosterone, in ethanol, final concentrations: 10E-11, 10E-10, 10E-9, 5*10E-9, 10E-8, 10E-7, 10E-6 mol/L

Antiandrogenic control: 5α-dihydrotestosterone combined with hydroxyflutamide, hydroxyflutamide was dissolved in DMSO, final concentrations: 5*10E-9 mol/L (5α-dihydrotestosterone) & 10E-5 mol/L (hydroxyflutamide)
The stability of the selected positive controls is well-defined under the chosen culture conditions since they are well established reference endocrine disruptors.

Results and discussion

Details on results:

Androgenicity:
An increase in the androgen receptor dependent enzyme expression (color development) was not observed.

Antiandrogenicity:
Due to inconclusive findings in the first experiment two further experiments were carried out. In the two further experiments, a concentration-related and reproducible inhibition of the androgenic effect compared to 5*10E-09 mol/L 5α-dihydrotestosterone (partly or total suppression of expected color development) was observed at concentrations from about 10E-05 mol/L onward.

Toxicity:
No cytotoxic effect (decrease of the yeast growth) was observed.

Solubility:
No test substance precipitation was found.

Any other information on results incl. tables

According to the results of this study, the test substance showed no androgenic activity in comparison to dihydrotestosterone in two experiments carried out independently. According to the results of this study, the test substance showed clear antiandrogenic activity in comparison to hydroxyflutamide in the 2nd and 3rd experiments carried out independently. Thus, under the experimental conditions chosen here, it is concluded that 4,4’-MDA did not exert androgenic effects while antiandrogenic effects were observed in the Yeast Androgen Screening (YAS) assay using the hAR yeast strain.