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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of the experimental phase: July 19, 2016; Termination of the experimental phase: August 12, 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): N,N‘-Methylenediacrylamide
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance):
- Substance type:
- Physical state: solid
- Analytical purity: No 'Certificate of Analysis' was available
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: LQ27A1677
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: No data on stability were available.
- Storage condition of test material: At +10°C to +25°C kept in a cool, dry and well-ventilated place.
- Other:

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified (EpiDermTM (EPI-200, Lot no. 23349) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava)
Source strain:
other: Reconstructed Human Epidermis Model
Details on animal used as source of test system:
other: Dulbecco's phosphate buffered saline (D-PBS)
Details on test system:

- Temperature (°C): 37°C
- Humidity (%): 95% relative humidity (RH) , 5% CO2


July 19, 2016 to August 12, 2016

Period of treatment: August 2016

Test system

Type of coverage:
other: The whole exposure period for the used EpiDermTM skin model was 60 minutes. The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
Preparation of test site:
other: 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
other: Dulbecco's phosphate buffered saline (D-PBS)
other: Three tissues were used for control groups
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 25 mg per skin model with a surface area of 0.63 cm2
- Concentration (if solution): 100% (only moistered with 25 µL D-PBS for better contact

- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): n.a.
- Lot/batch no. (if required): 1786985
- Purity: n.a.
Duration of treatment / exposure:
60 minutes
Observation period:
42 hours
Number of animals:
Three tissues were used treatment group
Details on study design:
- Area of exposure: skin model with a surface area of 0.63 cm2
- % coverage: uniformely covered
- Type of wrap if used: none

- Washing (if done): Dulbecco's phosphate buffered saline (D-PBS)
- Time after start of exposure: 60 minutes

mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS category 1 or 2 )
mean tissue viability > 50% non-irritant (NI)

Assay acceptability criteria
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5.
Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
Assay acceptance criterion 3: Standard deviation
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: tissue viability
> 50
Remarks on result:
Basis: mean. Time point: 6o minutes treatment and 42 hours incubation. Reversibility: other: n.a.. (migrated information)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Under the present test conditions, N,N‘-Methylenediacrylamide tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).
Executive summary:

The purpose of this study was to determine cytotoxic properties of N,N‘-Methylenediacrylamideto skin cells, which might lead to irritation ofhuman skin, by using an artificialthree-dimensionalmodel of human skin. TheEpiDermTMmodel was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assayand expressed as relative percentage of viability of the negative control-treated tissues.

N,N‘-Methylenediacrylamide was appliedtopically as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline. D-PBS was used as the negative control.5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to N,N‘-Methylenediacrylamide was 67.1% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%.N,N‘-Methylenediacrylamide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of the negative control of 3 tissues was 1.758 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.Theviability of cells treated with thepositive reference item, 5% SDS,was 5.1% of the negative control and fulfilled the acceptance criterion of ≤ 20%.

The standard deviation of all triplicates determined was below the limit of acceptance of 18%.Hence, allacceptance criteria were fulfilled.