1,3-bis(trimethylsilyl)urea

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 28 Jun 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2016

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2019

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(trimethylsilyl)urea

EC Number:

242-177-9

EC Name:

1,3-bis(trimethylsilyl)urea

Cas Number:

18297-63-7

Molecular formula:

C7H20N2OSi2

IUPAC Name:

1,3-bis(trimethylsilyl)urea

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation (Bratislava, Slovakia)

Source strain:

other: EpiDerm™ Skin Model (EPI-200 SCT)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Ashland, MA, USA)
- Date of initiation of testing: 26 Jun 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 ± 0.5 min at room temperature and 60 ± 5 min at 37 ± 1.5 °C
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with PBS to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL, 300 µL/well
- Incubation time: 3 h at 37 ± 1.5 °C
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.811 ± 0.096 (acceptance criteria 1.0 - 3.0)
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.43 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show colour changing or reducing capacity after 60 min MTT incubation.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Single experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and the viability after 1 hour exposure is less than 15%.
- The test item is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: The skin was moistened with 25 µL deionised water and 25 ± 2 mg (39.7 mg/cm^2) of the solid test item were applied onto the surface of the tissues.

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 ± 0.5 and 60 ± 5 min

Number of replicates:

Duplicates for each treatment and control group (3 ± 0.5 and 60 ± 5 min)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

98.45

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

116.82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test item did not show reducing capacity after 1 h incubation with MTT
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has demonstrated technical proficiency in the EpiDerm skin corrosion test by correct prediction of the corrosive potential of proficiency chemicals and by correct assignment of the chemicals into the sub-classes of corrosiveness as defined by the OECD guideline 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control was 1.755 ± 0.032 for the 3 min exposure and 1.49 ± 0.067 for the 1 h exposure and fell within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 15% compared to the negative control (8.26% for 1 hour exposure).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates was ≤ 30% (values between 1.2 - 6.9%).

Any other information on results incl. tables

Table 2: Results of the skin corrosion test for 3 minutes of exposure

Treatment Group	Tissue No.	Exposure Interval [min]	OD 570 nm			Mean OD of 3 wells blank corrected	Mean OD of 2 tissues blank corrected	Standard deviation of 2 tissues	Mean Rel. Viability [%]	CV [%]
			Well 1	Well 2	Well 3
Blank		3	0.039	0.039	0.039
Negative Control	1		1.845	1.803	1.812	1.781	1.755	0.032	100.00	2.0
	2		1.780	1.755	1.773	1.730
Positive Control	1		0.446	0.453	0.426	0.402	0.423	0.024	24.09	6.9
	2		0.477	0.486	0.485	0.443
Test Item	1		1.757	1.737	1.765	1.714	1.728	0.037	98.45	1.2
	2		1.724	1.797	1.824	1.742

Table 3: Results of the skin corrosion test for 60 minutes of exposure

Treatment Group	Tissue No.	Exposure Interval [min]	OD 570 nm			Mean OD of 3 wells blank corrected	Mean OD of 2 tissues blank corrected	Standard deviation of 2 tissues	Mean Rel. Viability [%]	CV [%]
			Well 1	Well 2	Well 3
Blank		60	0.039	0.039	0.039
Negative Control	1		1.483	1.500	1.456	1.440	1.490	0.067	100.00	4.7
	2		1.529	1.642	1.567	1.540
Positive Control	1		0.195	0.185	0.175	0.146	0.123	0.026	8.26	26.0
	2		0.143	0.139	0.137	0.100
Test Item	1		1.731	1.699	1.733	1.682	1.741	0.069	116.82	4.8
	2		1.804	1.854	1.860	1.800

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive according to Regulation (EC) No. 1272/2008.

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1, 1A, 1B/C) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.