1,3-bis(trimethylsilyl)urea

Endpoint summary

Description of key information

Skin sensitisation in vivo (OECD 429, LLNA): not sensitising

Skin sensitisation in vitro (OECD 442D, activation of keratinocytes): negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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]

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).

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

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).

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

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).

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 - 21 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An in vivo study was conducted because in vitro/in chemico test methods described under Annex VII, 8.3.1 of Regulation (EC) No. 1907/2006 are not applicable, or the results obtained from those studies are not adequate for classification and risk assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted in 2010

Deviations:

yes

Remarks:

relative humidity in the animal room was out of range for several hours (34 - 65% instead of 45 - 65%)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted in 2012

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc (AD Horst, The Netherlands)
- Females nulliparous and non-pregnant: yes
- Age at beginning of treatment: 8 - 12 weeks
- Weight at beginning of treatment: 18.8 ± 1.3 g (range: 16.7 - 21.1 g)
- Housing: Group housing in Makrolon type III cages (type II cages for preliminary toxicity study) with wire mesh top and granulated soft wood bedding.
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Air changes (per hr): at least 8 / h
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

other: cotton seed oil

Concentration:

Pre-test:10 and 25% (v/v)
Main study: 5, 10 and 25% (v/v)

No. of animals per dose:

1 (pre-test) and 5 (main study)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was found to be soluble in cotton seed oil as a 25% suspension. At higher concentrations or by the use of other vehicles, an applicable formulation of the test item was not achieved.
- Systemic toxicity: At 10 and 25% (w/v) the animals showed transiently hunched posture after the 2nd dose up to study Day 5. Decreased activity was noted after the 3rd dose but reversible by study Day 4 and at 25% the animal showed partially closed eyes on study Day 3 only. Wet fur, erythema of the scalp and scaly ears were noted for both animals at both dose levels until scheduled sacrifice on study Day 6.
- Ear thickness measurements: No increase in ear thickness of 25% or more at test item concentrations of 10 and 25% (v/v) was noted from study Days 1 – 3 and 1 - 6 and thus, the test substance was not considered irritating at these concentrations.
- Erythema scores: At 10 and 25% (w/v) the animals showed very slight to well-defined erythema (score 1 and 2) on the ears. The signs were observed until sacrifice on study Day 6. The findings were evaluated as non-excessive.

MAIN STUDY
Based on the pre-screen test results, test substance concentrations of 5, 10 and 25% (v/v) were chosen for the main study.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay. Cellular proliferation was determined by incorporation of 3H-methyl-thymidine (3HTdR)
- Criteria used to consider a positive response: The test item was considered positive in the LLNA if the incorporation of 3HTdR, indicated by stimulation index, was at least 3-fold when compared to control mice and if a dose-response relationship was observed.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in cotton seed oil. 25 µL/ear/day was spread over the entire dorsal surface (about 8 mm in diameter) of each ear once daily for three consecutive days.
Five days after the first topical application (Day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.9 µCi of 3H-methyl thymidine (equivalent to 79.4 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. About 5 hours after 3HTdR injection, the animals were euthanized with CO2, followed by harvesting of the lymph nodes and subsequent cervical dislocation.
The draining lymph nodes were pooled for each experimental group (8 nodes per group) and single cell suspensions of pooled lymph node cells were prepared. Two washing steps with PBS were performed, prior to re-suspension of the lymph node cells in 5% trichloroacetic acid and incubation at approx. 4 °C for 18 h for precipitation of macromolecules. Afterwards, the precipitates were re-suspended in 5% trichloroacetic acid and the level of 3HTdR was determined by use of a beta-scintillation counter measuring the number of radioactive disintegrations per minute (DPM).

CLINICAL SIGNS
All animals were observed daily, including pre- and post-dose observations on Days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

BODY WEIGHT
The body weights were recorded on Day 1 (prior to dosing) and prior to treatment with 3HTdR.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics. For body weights, mean values and standard deviations were calculated.

Positive control results:

A periodic positive control experiment using 5, 10 and 25% solutions of alpha-hexyl-cinnamaldehyde (v/v) in acetone:olive oil (4:1) was performed in Nov 2019. The SI values obtained were 1.68, 1.78 and 8.19 at 5, 10 and 25 %, demonstrating the validity of the test.

Key result

Parameter:

SI

Value:

0.89

Test group / Remarks:

5% formulation

Key result

Parameter:

SI

Value:

0.72

Test group / Remarks:

10% formulation

Key result

Parameter:

SI

Value:

0.61

Test group / Remarks:

25% formulation

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION:
The SI of the 5, 10 and 25% treatment group was 0.89, 0.72 and 0.61%, respectively.

EC3 CALCULATION: The EC3 value could not be calculated, since all S.I. values awere below the threshold value of 3.

CLINICAL OBSERVATIONS:
Mortality was not observed in the treatment or control groups and there were no signs of severe systemic toxicity. At 10 and 25%, hunched posture was transiently observed in 4/4 and 4/4 animals after the third dose administration, but fully reversible until study Day 4. At terminal sacrifice, all mice of the 5, 10 and 25% groups had scaly ears. The findings were considered to be without toxicological relevance.

LOCAL IRRITATION:
Very slight erythema (score 1) was noted in 4/4 and 4/4 animals of the 10 and 25% dose groups after the second dose administration. After the third dose administration, very slight erythema (score 1) was noted in 4/4 and 4/4 animals of the 5 and 10% groups and all mice of the 25% dose group showed well defined erythema (score 2).While the animals of the 5 and 10% dose groups were without any irritation effects by study Day 4, the animals of the 25% dose group still had very slight erythema on study Day 4. All effects were fully reversible by study Day 5.

BODY WEIGHTS:
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the control group over the same period.

Table 1: Results on stimulation indices

Test item concentration (%)	Measurement DPM	Calculation		Result
		number of lymph nodes	DPM per lymph node^#	S.I.
—	16	—	—	—
—	42	—	—	—
0	16456	8	2053.4	1.00
5	14703	8	1834.2	0.89
10	11933	8	1488.0	0.72
25	10124	8	1261.9	0.61
^#: Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Table 2: Results of the positive control (performed in Nov 2019)

Positive control cocentration (%)	Measurement DPM	Calculation		Result
		number of lymph nodes	DPM per lymph node^#	S.I.
—	10	—	—	—
—	10	—	—	—
0	6901	8	861.4	1.00
5	11583	8	1446.6	1.68
10	12260	8	1531.2	1.78
25	56477	8	7058.4	8.19
^#: Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

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 sensitising)

Additional information:

Skin sensitisation in vitro:

Inflammatory response in keratinocytes: KeratinoSens Assay

 

A KeratinoSensTM assay was performed in accordance with OECD guideline 442D and in compliance with GLP (Covance CRS Ltd., 2020) to evaluate the ability of N,N-Bis-(trimethylsilyl)urea to activate the antioxidant/electrophilic responsive element (ARE)-dependent pathway. The tests uses a transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element (the KeratinoSens^TM cell line).

Two independent experiments were performed using triplicate cultures per condition. The test item was dissolved in DMSO and the transgenic keratinocytes were exposed at 12 concentrations in the range of 0.24 – 500 µM. Solvent (DMSO, 1% in cultivation medium) and positive controls (cinnamic aldehyde, 4 – 64 µM in DMSO) were included in each experiment. After 48 ± 2 h of exposure at 37 ± 2 °C, luciferase activity was quantified and cytotoxicity was assessed by MTT assay.

The luminescence reading of the negative control was < 20 %, confirming the validity of the test. The positive control cinnamic aldehyde produced a statistically significant luciferase induction > 1.5 fold and the obtained EC_1.5 values in the two experiments were 21.23 and 15.19 µM, falling into the historical control range mean ± standard deviation generated by the testing facility. The acceptance criterion for an 2 – 4-fold induction at 64 µM for cinnamic aldehyde was not met in the first (1.89-fold induction), but in the second experiment (2.85-fold induction). However, as a clear dose-response relationship was observed and because all remaining acceptability criteria were met, the test was considered acceptable.

The test substance N,N-Bis-(trimethylsilyl)urea showed no cytotoxicity.The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values were calculated. The maximum luciferase induction (Imax ) obtained was 1.07 in the first and 1.27 in the second experiment. As the I_max for both assays was < 1.5 fold when compared to the solvent control, the EC_1.5 could not be calculated.

Based on the experimental findings and under the conditions of the test, N,N-Bis-(trimethylsilyl)urea gave a negative response in the KeratinoSens^TM 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.

 

In addition to the KeratinoSens assay, skin sensitisation testing was also initiated in chemico using a protein reactivity DRPA assay (no study report available). However, the substance rapidly hydrolyses in water, therefore the DPRA assay was considered not applicable and the testing for protein reactivity was not continued. Therefore, as in vitro/in chemico testing for skin sensitisation was not applicable and/or not adequate for classification and risk assessment of the test item, assessment of skin sensitisation was continued in vivo.

 

Skin sensitisation in vivo:

The skin sensitisation potential of N,N-Bis-(trimethylsilyl)urea was evaluated in a Local Lymph Node Assay (LLNA) that was performed according to OECD guideline 429 and in compliance with GLP (ICCR-Roßdorf GmbH, 2020b). Based on the results of a pre-test, in which test item formulations of 10 and 25% (v/v) did not show excessive local skin irritation or relevant signs of systemic toxicity, the main LLNA was performed with test item formulations of 5, 10 and 25% (v/v).

The test item was dissolved in cotton seed oil and 25 µL each were administered on three consecutive days to the dorsal surface of both ears of 4 female CBA/Ca mice. A similar constituted group of animals received the solvent (cotton seed oil) and served as control. Positive control animals administered 5, 10 and 25% of alpha-hexyl-cinnamaldehyde were included in a separate experiment that was performed several months before the main study with the test item. Five days after the first topical application of the test item, solvent or positive control (study Day 6), the mice were injected intravenously with ³H-methyl-thymidine (³HTdR). At terminal sacrifice, the auricular lymph nodes were harvested and the incorporation of ³HTdR was quantified by liquid scintillation as an indicator of cell proliferation. All animals were observed for clinical signs of systemic toxicity and local irritation effects were recorded. Individual body weights were measured prior to the first application and at study termination.

All animals of the 10 and 25% dose group showed hunched posture after the third dose administration, which was fully reversible by study Day 4. At terminal sacrifice, all animals of all test item-treated groups had scaly ears. The findings were considered to be without toxicological significance.

Very slight erythema (score 1) was noted in all animals of the 10 and 25% dose groups after the second dose administration. After the third dose administration, very slight erythema (score 1) was noted in all animals of the 5 and 10% groups. All mice of the 25% dose group showed well defined erythema (score 2) on study Day 3 and very slight erythema (score 1) on study Day 4. By Day 5, all animals of all groups fully recovered from any local irritation effects. The test item had no effect on body weight development during the study.

Stimulation indices (SI values), obtained for the test item were 0.89, 0.72 and 0.61% at 5, 10 and 25% (v/v), respectively. As the SI values were below the threshold of 3, no EC3 values were calculated. The experiment with the positive control alpha-hexyl-cinnamaldehyde gave a significant response (SI value 8.19) at 25%, confirming the validity of the protocol used for this study.

Under the conditions of the test, N,N-Bis-(trimethylsilyl)urea is not considered to have a skin sensitising potential.

 

Conclusion:

Based on the results of one in vivo study (LLNA), the test item N,N-Bis-(trimethylsilyl)urea is considered not to be a skin sensitiser. The result is supported by a KeratinoSens^TM in vitro assay, in which a negative result for skin sensitisation was obtained.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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