Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Research study not equivalent or similar to OECD guideline. Useful data for evaluation.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
In a compilation of 517 chemicals, that had been screened for estrogenic activity in vitro using a yeast two-hybrid assay, also 2,4-TDA is listed. Whereas 64 out of the 517 tested compounds were evaluated as positive, for 2,4-TDA no estrogenic activity was revealed.
GLP compliance:
not specified
Type of method:
in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No data.

Test animals

Species:
other: Yeast cells (Saccharomyces cerevisiae Y190)
Details on test animals and environmental conditions:
Not applicable.

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
other: DMSO or water
Details on exposure:
The yeast two-hybrid system based on the ligand-dependent interaction of nuclear hormone receptors with coactivators.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data.
Duration of treatment / exposure:
4 hours
Frequency of treatment:
Once
Duration of test:
Over 4 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
2.5 ul of 2,4-TDA
Basis:
no data
No. of animals per sex per dose:
Not applicable.
Details on study design:
The yeast two-hybrid assay system was used with the estrogen receptor, ERα, and the coactivator, TIF2. Two expression plasmids, pGBT9-ERLBD and pGAAD424-TIF-2, introduced into yeast cells (Saccharomyces cerevisiae Y190), which carry a β-galactosidase reporter gene and require tryptophan and leucine for growth. The cells were preincubated overnight at 30°C in SD medium free from tryptophan and leucine. The culture (250 μl) in a small test tube was then mixed with a DMSO solution (2.5 μl) of test chemical and incubated for 4 h at 30°C. After washing by centrifugation, the cells were digested enzymatically by incubation with 1 mg/ml Zymolyase 20T (200 μl) at 30°C for 15 min. The lysate was mixed with 4 mg/ml ONPG (40 μl)
and reacted until development of a yellow color (usually 30 min) before the reaction was stopped by the addition of 1 M Na2CO3 (100 μl). An aliquot (150 μl) was taken into each of 96 wells of a microplate. Absorbances at 420 and 550 nm were read on a microplate reader to estimate estrogenic activity.

The results were evaluated by relative activity, expressed as REC10 (10% relative effective concentration), that is the concentration of 2,4-TDA showing 10% of the agonist activity of 10–7 M E2, which is the optimum concentration for E2. When the activity of the test substance was higher than REC10 within the concentration tested, we judged the chemical as positive. When it was judged to be negative, we indicated the highest dose we tested.
Statistics:
No data

Results and discussion

Observed effects

For 2,4-TDA no estrogenic activity was revealed.

Applicant's summary and conclusion

Conclusions:
In a compilation of 517 chemicals, that had been screened for estrogenic activity in vitro using a yeast two-hybrid assay, also 2,4-TDA is listed (Nishihara et al., 2000). Whereas 64 out of the 517 tested compounds were evaluated as positive, for 2,4-TDA no estrogenic activity was revealed.
Executive summary:

In this yeast two-hybrid assay, 2,4 -TDA showed no estrogenic activity.