Tetravinylsilane

Endpoint summary

Administrative data

Description of key information

Two in vitro and one in vivo tests are available for the submission substance.

A direct peptide reactivity assay conducted according to OECD test guideline 442C and a KeratinoSens assay conducted according to OECD 442D with test item are available and report negative responses.

The LLNA test was conducted based OECD 429. Three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.7, 1.1 and 2.0, respectively.

Since there was no indication that the test item elicited a SI≥3 when tested up to 100%, the test item was considered not to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-09-13 to 2017-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

other: direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Lot number: 707001
Purity: 98.6%
Appearance: Colourless tranparent liquid
Storage conditions: Refrigerator (1-10°C) in an air-tight container

Details on the study design:

Cysteine peptide standard formulated in 100 mM phosphate buffer (pH 7.5) at 0.667 mM which was then diluted with acetonitrile to give 0.534 mM. Serial dilutions were performed with phosphate buffer/acetonitrile mixture (8/2 v/v) to give 0.267, 0.134, 0.0667, 0.0334 and 0.0167 mM.

Lysine peptide standard formulated in 100 mM ammonium acetate buffer (pH 10.2) at 0.667 mM which was then diluted with acetonitrile to give 0.534 mM. Serial dilutions were performed with ammonium acetate buffer/acetonitrile mixture (8/2 v/v) to give 0.267, 0.134, 0.0667, 0.0334 and 0.0167 mM.

The positive control solution was 100 mM cinnamaldehyde in acetonitrile.

The test substance solution was formulated at 100 mM in acetonitrile.

The high performace liquid chromatography (HLPC) instrument used L-2130 pumps, L-2200 autosampler, L-2400 UV-Vis detector, L-2300 column oven and EZChom Elite data processor.

The analytical conditions were:
Column: L-column2 ODS column (2.1 mm internal diameter x 100 mm; CERI)
Column oven temperature: 30°C
Mobile phase A: 0.1% trifluroacetic acid aqueous solution
Mobile phase B: 0.085% trifluoroacetic acid acetonitrile solution
Flow rate: 0.35 mL/min
Wavelength: 220 nm
Injection volume: 3 µL
Autosampler temperature: 25°C
Autosampler rinse solution: Acetonitrile/distilled water (1/1 v/v)

All samples were analysed using these HPLC conditions.

Calibration curves were prepared for each peptide. The coefficient of determination (r squared) were >0.990, therefore confirmed to meet the acceptance criteria.
Verification of suitability was conducted with 750 µL of the 0.667 mM peptide standard stock mixed with 250 µL of acetonitrile (Reference control A) which confirmed the mean concentrations of each peptide were 0.50 ± 0.05 mM. Samples were anlaysed in triplicate.
Verification of retention time (non-GLP) was conducted. Co-elution controls were prepared for each peptide. For the cysteine co-elution control, 750 µL was mixed with 200 µL of acetonitrile and 50 µL of test substance. For the lysine co-elution control, 750 µL of ammonium acetate buffer was mixed with 250 µ of test substance. The co-elution controls were allowed to stand at 25°C for 25 hours and then analysed. No test substance peaks were detected at the retention times of the peptides.

Reference control B was prepared in sextuplicate for each peptide by mixing 750 µL of 0.667 mM standard stock solution with 250 µL of acetonitrile.
Reference control C was prepared in triplicate in the same way.

The positive control reaction solutions were prepared in triplicate. For the cysteine peptide, 750 µL of 0.667 mM standard stock solution was mixed with 200 µL of acetonitrile and 50 µL of positive control solution. For the lysine peptide, 750 µL of 0.667 mM standard stock solution was mixed with 250 µL of positive control solution.
The test substance reaction solutions were prepared in triplicate. For the cysteine peptide, 750 µL of 0.667 mM standard stock solution was mixed with 200 µL of acetonitrile and 50 µL of test substance solution. For the lysine peptide, 750 µL of 0.667 mM standard stock solution was mixed with 250 µL of test substance solution. The reaction solutions were visually inspected at the time of preparation and 23 hours after preparation to confirm there was no suspension or precipitation.

Reference controls B and C, the positive control reaction solution and test substance reaction solution were analysed for each peptide. The analysis of the positive control reaction solutions and test substance reaction solutions were conducted after at least 24 hours following preparation.

The coefficients of variance for the reference controls met the acceptance criteria (<15.0%). The mean concentrations of reference control C were within 0.50 ± 0.05 mM for each peptide.

The peptide depletion was calculated as [1-(peptide peak area of each reaction solution/mean peptide peak area in reference control C)] x 100.
If this was negative, the value was taken as zero. The reactivity class was dependent on the mean value of the cysteine and lysine depletion.

Positive control results:

The mean cysteine peptide depletion was 76.3 ± 0.6%.
The mean lysine peptide depletion was 48.3 ± 4.8%.

Run / experiment:

other: 1

Parameter:

other: Mean cysteine depletion (%)

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 1

Parameter:

other: Mean lysine depletion (%)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: Mean cysteine and lysine depletion (%)

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria were met for the positive control. The mean depletion and the standard deviation were within the acceptable ranges.

Summary of results

Group		Cysteine depletion				Lysine depletion
		Individual	Mean	SD	CV	Individual	Mean	SD	CV
Test item	1	-2.5	0.4	0.6	150	-2.3	0.0	0.0	-
	2	1.1				-1.0
	3	-3.52				-5.1
Positive control	1	76.2	76.3	0.6	0.8%	53.8	48.3	4.8	9.9%
	2	75.8				46.1
	3	76.9				45.0

 

Interpretation of results:

other: negative

Conclusions:

The mean cysteine and lysine depletion was 0.2%. Based on this, the reactivity class of test item, was classified as 'no or minimal reactivity'. The skin sensitisation potential was predicted to be 'negative' in ths study.

Executive summary:

A GLP compliant study was conducted according to OECD test guideline 442C to predict the skin sensititisation of the test substance. The test substance was dissolved in acetonitrile at a concentration of 100 mM and mixed with cysteine peptide solution or lysine peptide solution. These reaction solutions, prepared in triplicate were incubated at 25°C for at least 24 hours. A positive control (cinnamaldehyde) was also included in the study. The reaction mixtures were analysed using high performance liquid chromatography (HPLC) and the peak area for each peptide was determined. The percent mean cysteine depletion and lysine depletion were determined and the mean percent cysteine and lysine depletion was calculated. The mean cysteine and lysine depletion was 0.2%, classifying the test substance as 'no or minimal reactivity' thereby the skin sensitisation potential was predicted as 'negative'.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2018-02-16 to 2018-03-30

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:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Appearance: Clear colourless liquid
Batch: 707001
Purity: 98.5%
Storage: At room temperature
Stable until: 30 July 2018 (expiry date)

Details on the study design:

- Test System:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

- Test Concentrations: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%).

- Experimental Design:
Cultures of 80-90% confluent KeratinoSens(TM) cells were sub-cultured into 96-well plates (10,000 cells/well). Cells were exposed to concentrations of the test material (dissolved in DMSO) or controls for 48 hours at 37±1.0 °C in the presence of 5% CO2. Following exposure, culture medium containing the test material was removed, mixed (1:1) with luciferase assay solution and added to the cells. Cells were shaken for 3 minutes and luciferase activity in the cell lysates measured on a plate reader. Exposures were made in triplicate and the experiment was repeated.

In parallel with the luciferase assay, an assessment of cytotoxicity was made in single replicates. Following the 48-hour exposure period, medium containing the test material was replaced with medium containing MTT. Cells were incubated with MTT for three hours and absorption measured using an automated plate reader.

Cells were also exposed to the positive control (ethylene dimethacrylate glycol) and negative (vehicle) control, DMSO. Blank wells were used to take into account background activity.

The following parameters were calculated:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability

Positive control results:

In Experiment 1 the positive control (ethylene dimethacrylate glycol) caused a concentration-related induction of the luciferase activity. The Imax was 2.31 and the EC1.5 was 100 μM.

In Experiment 2 The positive control (ethylene dimethacrylate glycol) caused a concentration-related induction of the luciferase activity. The Imax was 2.44 and the EC1.5 was 97 μM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.09

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
The luciferase activity induction obtained with the positive control (ethylene dimethacrylate glycol) was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 μM (100 μM and 97 μM in Experiments 1 and 2, respectively). A concentration-response was observed and the induction at 250 μM was higher than 2-fold (2.31 -fold and 2.44 -fold in Experiments 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.4% and 6.7% in Experiments 1 and 2, respectively). It is therefore concluded that the test conditions were adequate and that the test system functioned properly.

Experiment 1

No precipitation was observed at the start or end of the incubation period in the 96-well plates. The test item showed toxicity: the calculated IC30 was 327 μM and the calculated

IC50 was 376 μM. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with test item. The Imax was 1.09 and therefore no EC1.5 could be calculated.

Experiment 2

No precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed no cytotoxicity; the viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with test item. The Imax was 1.12 and therefore no EC1.5 could be calculated.

Summary of luminescence and cytotoxicity

Concentration (µm)		0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Luminescence	Exp 1	0.93	1.07	1.03	1.09	1.00	1.04	1.06	1.08	1.07	0.06	0.00	0.00
Viability (%)		109	103	92.9	95.4	92.2	89.8	89.6	92.3	101	-	-	-
Luminescence	Exp 2	1.00	1.04	1.03	0.98	0.95	0.90	0.94	1.00	0.97	0.96	1.07	1.12
Viability (%)		113	105	98.9	92.7	90.0	90.9	92.5	91.9	99.1	108	114	127

Overview 

		EC15 (µm)	Imax	IC30 (µm)	IC50 (µm)
Exp 1	Test item	-	1.09	327	376
Exp 2		-	1.12	-	-
Exp 1	Positive control	100	2.31	-	-
Exp 2		97	2.44	-	-

 

Interpretation of results:

GHS criteria not met

Conclusions:

In the first experiment, the test item showed toxicity (IC30 value of 327 μM and IC50 value of 376 μM). In the second experiment, the test item showed no toxicity (no IC30 and IC50 value). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.09-fold and 1.12-fold in Experiments 1 and 2 respectively. The test item is classified as negative in the KeratinoSens assay as negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Executive summary:

The skin sensitisation potential of test item was assessed in vitro in a KeratinoSens(TM) assay. The test item was dissolved in DMSO and tested at concentration up to 2000 μM. The assay was performed in triplicate and repeated in an independent experiment. Parallel cytotoxicity measurements were made in single replicates. Cells were also exposed to the positive control (ethylene dimethacrylate glycol) and negative (vehicle) control, DMSO.  Blank wells were used to take into account background activity. Both tests passed the acceptance criteria.

In the first experiment, the test item showed toxicity (IC30 value of 327 μM and IC50 value of 376 μM). In the second experiment, the test item showed no toxicity (no IC30 and IC50 value). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.09-fold and 1.12-fold in Experiments 1 and 2 respectively. The test item is classified as negative in the KeratinoSens assay as negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-05-14 to 2019-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch No.: 707001
Purity: 98.5%

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Inbred, SPF-Quality
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 19.8 to 24.0 g.
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities
- Diet: Pelleted rodent diet, ad libitum
- Water: Municipal tap-water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 42 to 56%.
- Air changes (per hr): Ten or greater
- Photoperiod: 12-hour light/12-hour dark cycle

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50, 100 %w/w

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
At both a 50% and 100% test item concentration, no signs of systemic toxicity were noted and very slight erythema and/or scaliness were observed between Days 1 and 3. Therefore the 100% concentration was selected as highest concentration for the main study.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Animals were assigned to the study at the discretion of the coordinating bio technician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
- Criteria used to consider a positive response: SI ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of Test Item:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
- Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity - Day 6:
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM)

OBSERVATIONS
- Mortality/Moribundity Checks: twice daily, in the morning and at the end of the working day.
- Clinical Observations: once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Body Weights: weighed individually on Day 1 (predose) and 6 (prior to necropsy).
- Irritation: once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing),

Positive control substance(s):

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

Positive control results:

An EC3 value of 16.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 14.3%.

Parameter:

SI

Value:

0.7

Test group / Remarks:

25% w/w group

Parameter:

SI

Value:

1.1

Test group / Remarks:

50 % w/w group

Parameter:

SI

Value:

2

Test group / Remarks:

100 % w/w group

Cellular proliferation data / Observations:

- Skin Reactions / Irritation:
The very slight erythema and/or scaliness noted for the test item treated animals between Days 1 and 4 was considered not to have a toxicologically significant effect on the activity of the nodes.
- Systemic Toxicity:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area:
The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals treated at 100% which were considered to be slightly enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements and SI Values:
Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 622, 937 and 1692 DPM, respectively. The mean DPM/animal value for the vehicle control group was 835 DPM.

Conclusions:

The SI values calculated for the test item concentrations 25, 50 and 100% were 0.7, 1.1 and 2.0, respectively. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, the test item was considered not to be a skin sensitizer.

Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice after three epidermal exposures of the animals based on OECD 429.

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)).

The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals treated at 100% which were considered to be slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 622, 937 and 1692 DPM, respectively. The mean DPM/animal value for the vehicle control group was 835 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.7, 1.1 and 2.0, respectively.

Since there was no indication that the test item elicited a SI≥3 when tested up to 100%, the test item was considered not to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Respiratory sensitisation:

Data lacking.

Skin sensitisation:

DPRA assay, OECD 442C: The test item shows low reactivity and therefore predicts a lack of sensitisation potential.

KeratinoSens assay, OECD 442D: The test item show a lack of activity and also predicts a lack of sensitisation potential.

LLNA, OECD 429: The SI values calculated for the test item concentrations 25, 50 and 100% were 0.7, 1.1 and 2.0, respectively.

Therefore in accordance with Regulation (EC) No. 1272/2008 (as amended by Regulation (EC) No. 286/2011) Table 3.4.2, and Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), this substance should not be classified as skin sensitisers based on the test data.