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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Estrogenic activities of chemicals related to food contact plastics and rubbers tested by the yeast two-hydrid assay.
Ogawa Y, Kawawamura Y, Wakui C, Mutsuga M, Nishimura T, and Tanamoto.
Bibliographic source:
Food Additives and Contaminants, (23)4:422-430.

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
4,4'-dithiodimorpholine induced by the S9-mix was tested for its estrogenicity activity using the yeast two-hybrid assay.
GLP compliance:
not specified
Type of method:
in vitro

Test material

Constituent 1
Chemical structure
Reference substance name:
Di(morpholin-4-yl) disulphide
EC Number:
EC Name:
Di(morpholin-4-yl) disulphide
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:

Administration / exposure

Route of administration:
Details on exposure:
DTDM were dissolved in DMSO at 10^-1 to 10^-5 mol/l (final concentration = 10^-3 to 10^-7 mol/l).
The concentration of DMSO was 1% in the assay, which did not inhibit the yeast growth.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
no data
Frequency of treatment:
no data
No. of animals per sex per dose:
no data
Control animals:
yes, concurrent vehicle
Details on study design:
The yeast two-hybrid cells were preincubated overnight at 30°C with vigorous shaking in a SD medium which was free from tryptophan and leucine. The culture was diluted with 4 volumes of the fresh SD medium and 250 µl of this solution put into a small test tube. The test chemical solution (2.5 µl) was added and incubated for 4h at 30°C. After incubation, 150 µl of the culture solution was placed into each of the 96 wells of a microplate and the absorbancy measured at 595 nm. The rest of the culture was centrifuged at 10,000 rpm for 7 min, alter which the supernatant was removed. The cells were enzymatically digested by incubation with 1 mg/l Zymolyase 20T (200 µl) at 30°C for 15 min. The cell lysate was mixed with 4 mg/ml ONPG (40 µl) and incubated at 30°C for exactly 30 min. The reaction was stopped by the addition of 1 mol/l Na2CO3 (100 µl). After centrifugation at 10,000 rpm for 5 min, the supernatant (150 µl) was placed into each well of a microplate. The absorbances at 420 and 570 nm were read using a microplate reader. The beta-galactosidase activity was expressed as the mean and standard deviation of the results from three separate test tubes.
Preparation of metabolites and their measurement of estrogenic activity :
To a tube containing 990µl of the S9-mix, 10µl of the test chemical solution (mainly 10^-1 to 10-5 mol/l ) was added, incubated at 37°C for 4h and then stored at -80°C until the yeast two-hybrid test was mn as metabolite solution. Each experiment was accompanied by trans-stylbene to confirm the metabolic activity. The yeast two-hybrid cells were pre-incubated overnight at 30°C with vigorous shaking in a SD medium free from tryptophan and leucine, then diluted with 1.5 volumes of fresh 2 x SD medium. In a small test tube, 125 µl of the cell solution and 125µl of the metabolite solution were mixed and then incubated at 30°C for 4h. Thereafter, the same procedure as the Measurement of estrogenic activity by yeast two-hybrid assay was carried out.


The results were evaluated on the basis of the relative activity, expressed as 10% relative effective concentration (REC10), which is the concentration of the test chemical showing 10% of the agonist activity of 10^-6 mol/l E2, the highest activity level of E2. When the activity of the test chemical was higher than the REC10 within the concentration range tested, the chemical was judged to be positive. When it was judged to be negative, more than the highest dose tested was indicated.
Positive control:
17beta-estradiol (E2)

Results and discussion

Details on results:
4,4'-dithiodimorpholine did not display any estrogenic activity.

Any other information on results incl. tables

Table : Estrogenic activities of DTDM



REC 10 (mol/l)

Parent compound




> 1.0 x 10^-3

> 5.0 x 10^-4

E2 (positive control)


3.4 x 10^-10 *


* = positive result

Applicant's summary and conclusion

DTDM did not show any estrogenicity.
Executive summary:

In this study, other chemicals related to food contact plastics and rubbers, and their metabolites induced by the S9-mixture were tested for their estrogenic activities using the yeast two-hybrid assay. DTDM did not show any estrogenicity.