1,3-bis(trimethylsilyl)urea

Endpoint summary

Description of key information

Skin corrosion (OECD 431): non corrosive

Skin irritation (OECD 439): not irritating

Serious eye damage / irritation (OECD 437): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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

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 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)

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

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%).

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

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jul - 23 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2019

Deviations:

yes

Remarks:

technical proficiency not shown

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

technical proficiency not shown

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2012

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories (Lyon, France)

Source strain:

other: EpiSkin™ Small / Human Epidermis (SM/13) reconstructed three-dimensional human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ Small / Human Epidermis (SM/13; SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 19-EKIN-034
- Shipping date: 20 Aug 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 15 min at 37 ± 1.5 °C
- Temperature of post-treatment incubation: 42 h at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the treatment interval the tissues were removed immediately from the 12-well plate and gently rinsed with PBS to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- 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 EpiSkin™ Small / Human Epidermis tissue was assessed via MTT test (IC50 determination): The determined IC50 values were 1.7 mg/mL and fitted to the specification 1.5 mg/mL ≤ IC50 ≥ 3 mg/mL.
- Morphology: The histology of tissues revealed a well-differentiated epidermis consisting of organised basal, spinous and granular layers, and a multi-layered stratum corneum.
- Contamination: In blood of the donors, the absence of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs were verified. In epidermal cells of the donors, the absence of bacteria, fungus and mycoplasma were verified.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A pre-experiment on assessment of colour interference and MTT interference was performed. The test item turned out to be not-interfering and not directly reducing MTT.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to the skin if the relative mean tissue viability of three individual tissues after 15 min of exposure to the test item and 42 h of post-incubation is less or equal to 50% of the mean viability of the negative controls.
- The test substance is considered non-irritant to the skin if the relative mean tissue viability of three individual tissues after 15 min of exposure to the test item and 42 h of post-incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: The skin was moistened with 5 µL deionised water and approximately 10 mg (26 mg/cm^2) of the solid test item were added on top of the skin tissues and possibly spread to match the surface.

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% in water

Duration of treatment / exposure:

15 min at room temperature

Duration of post-treatment incubation (if applicable):

42 h at 37 ± 1.5 °C

Number of replicates:

3 replicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

15 min exposure

Value:

101.7

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: Not included by the testing laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean absolute OD570 nm of the three negative control tissues was 0.791 ± 0.07 and fell within the acceptance limits of OECD 439 (lower acceptance limit ≥ 0.6 and upper acceptance limit ≤ 1.5).
- Acceptance criteria met for positive control: Yes, the mean relative tissue viability of the three positive control tissues was ≤ 40% (3.6 ± 1.2%) relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation calculated from individual % tissue viabilities of the three identically treated replicates was ≤ 18% (range 1.2 – 9.4).

Table 2: Results of the skin irritation test in vitro

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 wells	Mean OD of 2 wells blank corrected	Mean OD of 3 tissues blank corrected	SD of 3 tissues	Relative viability [%] Tissue 1, 2, 3	Mean Rel. Viability [%]	Standard deviation
Blank		0.038	0.038	0.038
Negative Control	1	0.949	0.864	0.907	0.868	0.791	0.07	109.7	100.0	9.4
	2	0.774	0.742	0.758	0.720			91
	3	0.834	0.813	0.823	0.785			99.3
Positive Control	1	0.079	0.074	0.077	0.038	0.029	0.01	4.8	3.6	1.2
	2	0.059	0.056	0.057	0.019			2.4
	3	0.067	0.066	0.067	0.028			3.6
Test Item	1	0.889	0.841	0.865	0.827	0.805	0.04	104.5	101.7	5.1
	2	0.883	0.854	0.868	0.830			104.9
	3	0.806	0.787	0.796	0.758			95.8

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jul 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2017

Deviations:

yes

Remarks:

technical proficiency not shown; the positive control was 10% (w/v) Benzalkonium chloride in saline since the laboratory historical control data was established with this chemical

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

technical proficiency not shown; the positive control was 10% (w/v) Benzalkonium chloride in saline since the laboratory historical control data was established with this chemical.

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

adopted in 2010

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006.

Version / remarks:

adopted in 2017

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, Aschaffenburg, Germany
- Characteristics of donor animals: 14 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin / streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes and directly used in the BCOP test on the same day.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin / streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin)

Vehicle:

physiological saline

Remarks:

20% (w/v) suspension

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20% (w/v) in saline

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 10% (w/v) in saline

Duration of treatment / exposure:

240 min at 32 ± 1 °C

Number of animals or in vitro replicates:

Three corneas were used for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin). The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Following equilibration, an initial opacity reading was performed to determine the basal opacity (t0). Only corneas with a value of the basal opacity < 7 were used.

NUMBER OF REPLICATES
Sets of three corneas were used for treatment with the test item and for the negative and positive controls, respectively.

TREATMENT METHOD: Open chamber method
Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O- ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed and the basal opacity was determined (t0). For treatment, the anterior compartment received the test item suspension or the negative or positive controls at a volume of 750 µL each on the surface of the corneas. The corneas were incubated in a horizontal position at 32 ± 1 °C in the waterbath for 240 min.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of the test item the corneae were given a final rinse with cMEM without phenol red.

- POST-EXPOSURE INCUBATION: Not performed

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Following exposure, fresh cMEM was added into the anterior compartment and the opacity determinations were performed. Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax® Molecular Devices). The optical density at 490 nm (OD490) of each sampling tube was determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

240 min exposure

Value:

1.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not included by the testing laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses result in opacity (0.33) and permeability values (0.060) that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value (90.93 ± 6.32) which fell within two standard deviations of the current historical mean.

Table 2: Results of the BCOP test after 240 min of exposure

Test Group	Opacity value: Difference (t240 - t0) opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS ± SD	Evaluation
	Individual	Mean	Individual	Mean
Negative Control	1.0	0.33	0.062	0.06	1.93	1.24 ± 0.60	No Category
	0.0		0.060		0.90
	0.0		0.059		0.89
Positive Control	83.67*		0.381*		89.38	90.93 ± 6.32	Category 1
	79.67*		0.391*		85.53
	91.67*		0.415*		97.89
Test item	1.67*		0.00**		1.67	1.04 ± 0.54	No Category
	0.67*		0.00**		0.67
	0.67*		0.009*		0.80

* corrected values

** value was set to zero since the calculated value was negative

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

CLP: Not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion in vitro

The human Skin Model Test with EpiDerm^TM tissue models was conducted to assess the corrosive potential of the test item in human keratinocytes according to OECD guideline 431 and in compliance with GLP (Envigo CRS GmbH, 2019a).

The test item passed the colour interference and MTT reduction test and turned out to be non-interfering and non-reducing. Duplicate tissues of the human skin model EpiDerm^TM were each exposed to the test item, the negative control (deionized water) or the positive control (8.0 N KOH) for 3 ± 0.5 min (room temperature) and for 60 ± 5 min (37 ± 1.5 °C). 25 ± 2 mg of the test item (39.7 mg/cm²) or 50 µL of the negative or of the positive control were applied onto the surface of the skin tissues. After exposure, the tissues were gently rinsed with PBS to remove any residual test material and cytotoxicity was evaluated by MTT reduction.

Negative control absorbance values were within the range of historical control data and the positive control induced a decrease in viability as compared to the negative control (24.09% after 3 min and 8.26% after 60 min exposure), thus confirming the quality of the tissues and matching the acceptability criteria for the study. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 98.45% and 116.82%, respectively.

Under the conditions of the study, the test item is not considered to be corrosive to human skin.

 

Skin irritation in vitro

N,N-Bis-(trimethylsilyl)urea was investigated for its skin irritation potential in epidermal keratinocytes in vitro using reconstructed human epidermis. The test was conducted with EpiSkin™ Small / Human Epidermis according to OECD guideline 439 and in compliance with GLP (ICCR-Roßdorf GmbH, 2019).

Approximately 10 mg of the test item (26 mg/cm²) or 10 µL of the negative control (phosphate buffered saline, PBS) and the positive control (5% sodium dodecyl sulfate, SDS) were applied topically to the surface of the tissues and incubated for 15 min at room temperature. The exposure was followed by a 42 h post-incubation period at 37 ± 1.5 °C. After the post-exposure period, cytotoxicity was evaluated by MTT reduction.

The test item passed the colour interference and MTT reduction test and turned out to be non-interfering and non-reducing.

When compared to the mean relative tissue viability of the negative control, the mean relative tissue viability of N,N-Bis-(trimethylsilyl)urea was 101.7% after 15 min of exposure. This value is above the threshold for irritancy of ≤ 50%. All acceptability criteria for the negative and the positive controls were met and the standard deviation value of the percentage viability of three tissues treated identically was < 10 %, thus demonstrating the functionality of the test.

Under the conditions of the test, the test item is considered to be non-irritant to the skin.

 

Eye irritation in vitro

The eye irritating potential of N,N-Bis-(trimethylsilyl)urea was investigated using a bovine corneal opacity and permeability (BCOP) test in vitro. The BCOP test was conducted according to OECD guideline 437 and in compliance with GLP (Envigo CRS GmbH, 2019b). The test item was prepared as 20% (w/v) formulation in physiological saline. After an initial opacity reading, 750 µL of test substance formulation, negative control (physiological saline) or positive control (benzalkonium chloride (10% w/v) in physiological saline) were applied onto the epithelium of each three corneas. The tissues were incubated for 240 min at 32 ± 1 °C and rinsed off after exposure. Afterwards, a second opacity reading was performed. In addition, permeability of the corneas was evaluated by measuring the transfer of sodium fluorescein after incubation for 90 ± 5 min at 32 ± 1 °C.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory's historical control range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 90.93 ± 6.32 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Relative to the negative control, the calculated mean in vitro irritancy score (IVIS) of N,N-Bis-(trimethylsilyl)urea was 1.04 ± 0.54. Based on the experimental findings and under the conditions of the test, the test item is not considered irritant to the eye.

 

Respiratory irritation

In an acute inhalation toxicity study conducted according to OECD guideline 433 (Covance Laboratories Ltd., 2020a), Wistar rats were exposed to aerosols of the test item by nose-only application. Based on the results of a preliminary range-finding study, a group of 5 male rats was exposed for 4 hours to an aerosol concentration of 5.30 ± 0.225 mg/L air. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) was 2.2 ± 2.64 µm. During exposure and thereafter, the animals were observed for clinical signs of toxicity and mortality. During a 14-days observation period, the animals were inspected for general health and mortality and individual body weights were recorded on Day 1 (prior to dosing) and on Days 2, 4, 8 and 15. At terminal sacrifice, all animals were subjected to gross macroscopical examination.

For details of systemic toxicity, please refer to section 7.2 Acute toxicity. In the study, no local effects of respiratory irritation were observed in the animals and no irritation findings were observed at macroscopic examination. Based on the experimental findings, the test item is considered not to be a respiratory irritant.

Justification for classification or non-classification

The available data on skin irritation/corrosion, eye irritation/corrosion and respiratory irritation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.