Registration Dossier

Diss Factsheets

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.09.2012 - 07.09.2012
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6(1H,3H,5H)-Pyrimidinetrione, 5,5'-(1,2-diazenediyl)bis-
Cas Number:
Molecular formula:
2,4,6(1H,3H,5H)-Pyrimidinetrione, 5,5'-(1,2-diazenediyl)bis-
Test material form:
solid: crystalline
Details on test material:
Red solid powder

Test animals

other: in vitro - tissues

Test system

Details on study design:
Principle:The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide], present in cell mitochondria, into a blue formazal salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed tochemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential. Evaluation is determined by measuring of optical density (OD) of the formazan, extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean OD value of the negative control tissues. Skin irritation potential of the test material is predicted if the remaining relative cell viability is below 50 %.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: average viability
Run / experiment:
> 1.689 - < 1.727
Remarks on result:
Time point: 45 h. Reversibility: other: not relevant. Remarks: average viability = 88.8%. (migrated information)

Any other information on results incl. tables

MTT test

The test substance (25 mg) was placed directly atop to the tissue previously moistened with 25 pl of PBS. The material was then spread on the tissue surface. Tissues were exposed to the test chemical for 1 hour. After exposition, tissues were thoroughly rinsed and blotted to remove the test substance. Then, tissues were let to post-incubate for 24 (medium exchange) + 18 hours (37+1oC,1LIYI CO2, moistened).

After exposition, tissues were slightly coloured yellow; the yeliow colour was not observed after post-incubation.

Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. ODszo measuring was performed after 2-hour extraction with shaking. Results are given in the Table.

treatment parameter OD570 mean SD average viability
(% NC)
1 2 3
OD570 1.946 1.975 2.036 1.986 0.038  
viability 98.00 99.46 102.53 100.0 1.89 100.0
OD570 1.689 1.874 1.727 1.763 0.080  
viability 85.06 94.38 86.97 88.8 4.2 88.8
(5% SDS)
OD570 0.217 0.13 0.188 0.178 0.036  
viability 10.93 6.55 9.47 8.98 1.82 8.98
NC  negative control
PC  positive control
Cl  test substance
mean  arithmetic mean
SD  standard deviation calculated from individualyo tissue viabilities
viability (%) viability of single tissues compared with negative control

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
Under the above-described experimental design, average viability of tissues treated by the test substance was 88.8 % of negative control average value, i.e. viability was >50 %.The effect of test substance was negative in EpiDerm'* model.