1,3-bis(trimethylsilyl)urea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Oct - 07 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

acceptance criteria of the positive control not met in the first experiment (1.89 fold induction instead of 2 - 8 fold induction), technical proficiency not shown

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines and Healthcare products Regulatory Agency, UK

Type of study:

activation of keratinocytes

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

Details on the study design:

TEST METHOD
The in vitro KeratinoSensTM assay enables the detection of the skin sensitizing potential of a test item by analysing the activation of keratinocytes. This activation step represents the second molecular key event of the adverse outcome pathway, which is the induction of cyto-protective signaling pathways in keratinocytes in response to electrophilic test chemicals. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE) Nrf2-dependent pathway in the transgenic KeratinoSensTM cell line, which stably expresses the ARE-Nrf2-dependent luciferase gene. The Nrf2-dependent induction of this reporter gene is analysed upon exposure to test chemicals. Luminescence detection in the cell lysate after 48 ± 2 h of exposure at 37 ± 2 °C indicates luciferase induction and allows the discrimination between skin sensitisers and non-sensitisers.

TEST CELL LINE: KeratinoSensTM
- Cell type: HaCaT cells (human keratinocytes)
- Source: Givaudan (Dubendorf, Switzerland)
- Passage number: The passages of KeratinoSens™ cells were limited to 25 passages from frozen stock.

CELL CULTURE:
MEDIA:
Basic medium: 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM),
supplemented with 50 mL foetal bovine serum (FBS)
Maintenance medium: 500 mL DMEM, supplemented with 50 mL FBS and 5.5 mL geneticin
Exposure medium: 495 mL DMEM, supplemented with 5.0 mL FBS

MAINTAINANCE:
The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Cells were sub-cultured upon reaching 80-90% confluency. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.

CONCENTRATIONS:
0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 µM

CONTROLS:
VEHICLE CONTROL: Dimethyl sulfoxide
Final concentration in the exposure medium: 1% (v/v)

POSITIVE CONTROL: Cinnamic aldehyde (Sigma)
Solvent: DMSO
Concentration range: 4 – 64 µM

NUMBER OF REPLICATIONS: triplicates in two independent experiments

EXPERIMENTAL PROCEDURE:
Cells were seeded 1 x 10E5 cells / well in 96-well flat-bottomed microtitre plates in basic medium. Three plates were used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. After incubation for 24 ± 2 h, the medium was removed, replaced with antibiotic-free exposure medium and the test and control items were applied. The cells were exposed for 48 ± 2 h at 37 ± 2 °C. After the exposure period, luciferase activity was evaluated by luminescence measurement and cytotoxicity was assessed using the MTT viability assay.

LUCIFERASE ASSAY:
- Assay: Steady-Glo Luciferase Assay (Promega)
- Incubation time: The plates were shaken on a plate shaker for at least 5 min until the cells had lysed. Prior to measurement, the plates were adapted for 1 min in the dark.
- Luminometer: SpectraMax L luminometer

MTT VIABILITY ASSAY:
- MTT concentration: 5 mg/mL
- Incubation time: 4 h ± 10 min at 37 ± 2 °C
- Spectrophotometer: Plate Reader
- Wavelength: 540 nm

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

Cinnamic aldehyde (CA) was tested as positive control in a concentration range of 4 – 64 µM. In both experiments, luciferase activity increased dose-dependently with statistical significance. At 64 µM, the induction was 1.89 and 2.85, respectively. Thus, the acceptability criterion “average induction of positive control at 64 µM between 2 – 8 was not fulfilled in the first experiment. The EC1.5 was well within two standard deviations of the historical mean (21.23 µM in the first and 15.19 µM in the second experiment).

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

other: 48 h exposure, first experiment

Parameter:

other: maximum fold luciferase activity induction

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Remarks:

fold induction at 64 µM not 2 - 8 but 1.89-fold

Remarks on result:

other: fold induction for luciferase activity: 1.07

Results 2

Key result

Group:

test chemical

Run / experiment:

other: 48 h exposure, second experiment

Parameter:

other: maximum fold luciferase activity induction

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: fold induction for luciferase activity: 1.27

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The average induction in the three replicates for cinnamic aldehyde at 64 µM was not between 2 – 8 in the first experiment (1.89 fold induction only). In the second experiment, the average induction was 2.85 and met the acceptance criteria. As a clear dose-response was observed in both experiments, the result of the first test was nevertheless considered acceptable. In addition, all other acceptability criteria were fulfilled: The luciferase activity induction obtained with the positive control cinammic aldehyde was statistically significant above the threshold of 1.5-fold when compared to the solvent control in at least one concentration (1.89 and 1.89 at 32 and 64 µM in the first experiment and 1.54, 1.90 and 2.85 at 16, 32 and 64 µM in the second experiment, respectively). In addition, the EC1.5 of the positive control were 21.23 and 15.19 µM in the first and in the second experiment and fell within two standard deviations of the historical mean of the testing facility (-3.00 – 29.27 µM).
- Acceptance criteria met for variability between replicate measurements of the solvent control: The average coefficient of variation of the luminescence reading for the solvent control DMSO was below 20% in each repetition (12.1%, 16.4% in experiments 1 and 2, respectively).

Any other information on results incl. tables

No cytotoxicity was observed. The cellular viability did not fall below 72.56% in first and 92.20% in second experiment, and therefore the IC30 and IC50 could not be calculated.

Table 1: Results of the KeratinoSens assay with the test item

Test item conc. (µM)	First experiment				Second experiment
	Mean fold induction	Statistically significant	Viability (%)	I-max	Mean fold induction	Statistically significant	Viability (%)	I-max
0.24	1.07	No	133.55	1.07	0.75	No	101.72	1.27
0.49	0.84	No	114.15		0.86	No	106.44
0.98	0.82	No	117.14		0.92	No	104.84
1.95	0.84	No	113.86		0.93	No	106.36
3.91	0.89	No	114.15		0.98	No	101.16
7.81	0.80	No	72.56		1.27	No	98.44
15.63	0.91	No	108.85		0.93	No	92.20
31.25	0.90	No	98.62		1.01	No	94.36
62.5	0.86	No	103.54		1.05	No	97.80
125	0.74	No	108.36		1.01	No	97.72
250	0.67	No	104.12		0.98	No	103.80
500	0.60	No	101.42		0.92	No	107.16

Table 2: Results of the KeratinoSens assay with the positive control

Positive control conc. (µM)	First experiment					Second experiment
	Mean fold induction	Statistically significant	Viability (%)	I-max	EC1.5(µM)	Mean fold induction	Statistically significant	Viability (%)	I-max	EC1.5(µM)
4	1.24	No	99.58	1.89	21.23	1.12	No	98.60	2.85	15.19
8	1.33	No	89.06			1.18	No	106.76
16	1.31	No	98.04			1.54	Yes	109.72
32	1.89	Yes	98.52			1.90	Yes	102.84
64	1.89	Yes	93.12			2.85	Yes	116.67

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event “keratinocyte activation / inflammatory response”

Conclusions:

Based on the experimental findings and under the conditions of the test, the test item gave a negative response in the KeratinoSensTM assay. However, this was obtained with concentrations < 1000 µM and did not reach cytotoxicity (< 70% cell viability) at the highest tested concentration. Therefore, no conclusion on skin sensitisation is possible.
There is regulatory acceptance in the EU for the application of the KeratinoSens assay to address key event 2: activation of keratinocytes / inflammatory response in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance was negative for keratinocyte activation in two independent experiments. The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).