Bis(dimethylamino)methylvinylsilane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-07-28 to 2021-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethylamino)methylvinylsilane

EC Number:

236-437-0

EC Name:

Bis(dimethylamino)methylvinylsilane

Cas Number:

13368-45-1

Molecular formula:

C7H18N2Si

IUPAC Name:

N,N,N',N',1-pentamethyl-1-vinylsilanediamine

Test material form:

liquid

Details on test material:

Bis(dimethylamino)vinylmethylsilane, Batch: 9092008001

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDerm™ tissues were provided as kits (MatTek)

Justification for test system used:

The EpiDerm™ Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

Upon receipt of the EpiDerm™, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37 +/- 1 °C, 5.0% CO2 / 95% air.

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.
After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.

60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert: thus, the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min. Per each tissue 3 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

Further Reagents:
MTT solution
- MTT stock solution: 5 mg/mL MTT (VWR; Lot 20B2456894) in PBS
- MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)

Isopropanol

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance 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%.

Test Acceptance Criteria:
The test meets acceptance criteria if:
- mean absolute OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period is ≤ 15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied undiluted. 50 μL of the test item was dispensed directly atop the EpiDerm™ tissue.

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure (NSMTT corrected)

Value:

18.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure (NSMTT corrected)

Value:

13.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Pre-Experiments:
The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple.
For quantitative correction of results, the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, two tissues per treatment period were treated with the test item (KT) or left untreated (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) per treatment period according to the following formula:

NSMTT (3min) = [(ODKT - ODKU)/ODNC] * 100 = [(0.169 - 0.149)/ 1.777] *100 = 1.1%
NSMTT (60 min) = [(ODKT - ODKU)/ODNC] * 100 = [(0.114 - 0.137)/ 1.748] *100 = -1.3%

NSMTT was ≤ 30% relative to the negative control of living epidermis. In the 3 min experiment NSMTT was 1.1%, in the 60 min experiment -1.3%. This means that the test item was washed away almost completely before the addition of the MTT solution. The true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected for each treatment period according to the following formula:

TODTT (3 min) = ODTM - (ODKT - ODKU) = 0.350 – (0.169 - 0.149) = 0.331
TODTT (60 min) = ODTM - (ODKT - ODKU) = 0.214 – (0.114 - 0.137) = 0.236

The mixture of 50 μL test item per 300 μL Aqua dest. and per 90 μL isopropanol showed no colouring as compared to the solvent. Therefore, NSC equalled 0%.

The test item showed non-specific reduction of MTT, but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false negative results were necessary.

Applicant's summary and conclusion

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 3 min treatment was decreased below 25%. The test item is therefore classified as “corrosive“ in accordance with optional UN GHS sub-category 1A.

Executive summary:

In the present study the skin corrosivity potential of Bis(dimethylamino)methylvinylsilane was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis, the cytotoxic effects of the test item on EpiDerm™, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.
The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Therefore, the results were quantitatively corrected for the non-specific reduction of MTT (NSMTT) of the test substance.

The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 25% (18.6%, NSMTT-corrected) after 3 min treatment.