N,N'-methylenebis[methacrylamide]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-14 to 2017-04-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted July 28, 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted July 06, 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-methylenebis[methacrylamide]

EC Number:

219-102-3

EC Name:

N,N'-methylenebis[methacrylamide]

Cas Number:

2359-15-1

Molecular formula:

C9H14N2O2

IUPAC Name:

N,N'-methylenebis(2-methylacrylamide)

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis. This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2. The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes.

Vehicle:

other: The skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline for better contact of the test item to the skin.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model (MatTek)
- Tissue batch number: 25803
- Production date: 2017-04-05
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5 % CO2 and 95 % relative humidity
- Temperature of post-treatment incubation: 37 °C, 5 % CO2 and 95 % relative humidity
REMOVAL OF TEST MATERIAL AND CONTROLS
- the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (DPBS)
- Observable damage in the tissue due to washing: not reported
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2, Tecan Deutschland GmbH, 74564 Crailsheim, Germany
- Wavelength: 540 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: according to the manufacturer: MTT QC assay, 4 hours, n=3. Acceptance criteria: OD (540-570 nm) 1.0-3.0. Result: 1.7 ± 0.105. QA statement: pass.
- Barrier function: according to the manufacturer: ET-50 assay, 100 µL 1 % Triton X-100, 4 time-points, n=3, MTT assay. Acceptance criteria: ET-50 4.77-8.72 hours. Result: 5.36 hours. QA statement: pass.
- Morphology: Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers. Results obtained with this lot conform to the requirements of the OECD TG 431 and 439.
- Contamination: sterile; no contamination.
NUMBER OF REPLICATE TISSUES: 3
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg / 0.63 cm²
- Concentration: neat, but the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline for better contact of the test item to the skin.
NEGATIVE CONTROL
- Amount applied: 30 µL Dulbecco’s phosphate buffered saline (DPBS)
POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 % aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main experiment

Value:

ca. 118.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: not observed
- Colour interference with MTT: not observed
DEMONSTRATION OF TECHNICAL PROFICIENCY: The controls confirmed the validity of the study.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.418.
- Acceptance criteria met for positive control: yes. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %. The viability of cells treated with the positive reference item was 6.1 % of the negative control.
- Acceptance criteria met for variability between replicate measurements: yes. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18 %. The standard deviation determined for all triplicates was below the limit of acceptance of 18 %.

Any other information on results incl. tables

Test for interference of chemicals with MTT

No discolouration of the test item was noted neither in sterile water nor isopropanol.

No change of colour in MTT solution was noted.

Results

The mean viability of cells exposed to the test item was 118.7 % 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 %. The test item was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.418 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5 % SDS, was 6.1 % of the negative control and fulfilled the acceptance criterion of ≤ 20 %. The standard deviation determined for all triplicates was below the limit of acceptance of 18 %. Hence, all acceptance criteria required were fulfilled.

Table 1: Summarized Results of in vitro Skin Irritation. CV: coefficient of variation. SDS: sodium dodecyl sulphate. DPBS: Dulbecco's phosphate buffered saline.

	Optical density [OD540] mean tissue (n=3)	Mean viability (n=3) [%]	CV [%]
DPBS (negative control)	1.418	100.0	6.6
Test item	1.684	118.7	5.8
5% SDS (positive control)	0.087	6.1	5.3

Table 2: Individual values of in vitro Skin Irritation. CV: coefficient of variation. SDS: sodium dodecyl sulphate. DPBS: Dulbecco's phosphate buffered saline.

	DPBS (negative control)		Test item		5 % SDS (positive control)
replicates	Optical density [OD540]	Mean viability [%]	Optical density [OD540]	Mean viability [%]	Optical density [OD540]	Mean viability [%]
Tissue 1	1.509		1.866		0.087
	1.497		1.543		0.096
Mean (percent difference)	1.503 (0.8)	106.0	1.705 (18.9)	120.2	0.092 (9.8)	6.5
Tissue 2	1.307		1.504		0.079
	1.331		1.649		0.086
Mean (percent difference)	1.319 (1.8)	93.0	1.577 (9.2)		0.083 (8.5)	5.9
Tissue 3	1.377		1.634		0.083
	1.484		1.903		0.087
Mean (percent difference)	1.431 (7.5)	100.9	1.769 (15.2)	124.8	0.085 (4.7)	6.0
Mean 3 tissues (CV %)	1.418 (6.5)	100.0 (6.6)	1.684 (5.8)	118.7 (5.8)	0.087 (5.4)	6.1 (5.3)

Table 3: Historical data of negative and positive controls (Most recent background data from GLP studies of the years 2013 – 2016 (n = 18). Summary of EpiDerm Skin Irritation tested in independent lots of EpiDerm. If tissue viability is ≤ 50 % the test material is labelled as an irritant; if viable > 50 %, the test material is labelled as a non-irritant.

#1 Unqualified results = if the mean OD of the NC tissues is < 1.0 or > 2.5; if difference in viability for duplicate tissues > 18 %

#2 Unqualified results = viability > 20 %

CV: coefficient of variation

SDS: Sodium dodecyl sulphate

DPBS: Dulbecco's Phosphate-Buffered Saline

Material	Average OD (mean CV %)	Average Viability % (mean CV %)	Range		Irritant (I) / Non-irritant  (NI)	No. of  Unqualified Experiments
			Viability [%]	CV [%]
Negative control (DPBS)	1.900 (4.5 ± 2.2)	100 (4.5 ± 2.3)	88.7-108.9	1.5-10.3	NI	0^#1
Positive control  (SDS 5 %)	0.080 (8.1 ± 4.3)	4.3 (4.4 ± 1.8)	1.2-7.4	1.8-14.4	I	0^#2

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was non-cytotoxic after an exposure time of 60 minutes and a 42-hour post-treatment incubation period in an artificial three-dimensional model of human skin. The test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).

Executive summary:

In this study according to OECD guideline 439, the potential of N,N'-Methylenebis[methacrylamide] to induce skin irritation was analysed by using a three-dimensional human epidermis model (RhE). The test substance was applied topically for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

All controls confirmed the validity of the study. N,N'-Methylenebis[methacrylamide] showed no irritant effects. The mean relative tissue viability (% negative control) was >50 % (118.7 %) after 60 min treatment and 42 h post-incubation.

Hence, N,N'-methylenebis[methacrylamide] is classified as “non-irritant” in accordance with EU GHS “No Category”.