Bis(dimethylamino)methylvinylsilane

Endpoint summary

Description of key information

The test item was tested for skin corrosion and eye irritation according to OECD TG 431 and OECD TG 437. Both studies were carried out under GLP. The test item is classified as “corrosive“ in accordance with optional UN GHS sub-category 1A. Additionally, the test item is classified into eye irritant UN GHS Category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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

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:

2020

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir Vion Beef B.V., Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

appr. 750 µL

Duration of treatment / exposure:

10 mins

Duration of post- treatment incubation (in vitro):

2 hrs

Number of animals or in vitro replicates:

3

Details on study design:

The eyes were carefully examined for defects and any defective eyes were discarded.

The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.


Calibration of the Opacitometer

The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 90-100 lux. The calibration procedure was performed before the test and is documented in the raw data.


Treatment of the Corneas

After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 µL of the control substance was introduced into the anterior chamber. Due to high viscosity of the test item after contact to air during application, approximately 750 µL of the test substance was applied directly, completely covering the cornea, by removing the window-locking ring and glass window prior to treatment. After 10 minutes incubation at 32±1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed after 2 hours incubation at 32±1 °C. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32±1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Irritation parameter:

in vitro irritation score

Value:

319.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Evaluation of Results
The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:

𝑂𝑝𝑎𝑐𝑖𝑡𝑦= [(𝐼₀/𝐼)−𝑏]/𝑎

with a = 0.025 and b = 0.9894

The value I₀ is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is re-evaluated periodically.
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the Table:

IVIS	UN GHS
≤ 3	no category
>3; ≤55	no stand-alone prediction can be made
>55	Category 1

An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose, further testing with another suitable method is required.

 

Due to reaction (hydrolysis) of the test material after contact to air, the test item became more viscous so that a glass pipette instead of a steel canule attached to a scaled syringe had to be used for application. Thus, the application volume of 750 μl was approximated, taking care to cover the cornea completely with the test item as requested by guideline OECD 437, 2020.
This deviation did not influence the quality or integrity of the present study.

 

The results tables of the study are attached as pdf.

Conclusions:

According to the evaluation criteria the test item Bis(dimethylamino)methylvinylsilane is classified into UN GHS Category 1.

Executive summary:

The eye irritancy potential of Bis(dimethylamino)methylvinylsilane was investigated in the bovine corneal opacity and permeability assay. Due to reaction (hydrolysis) of the test material after contact to air, the test item changed from a translucent non-viscous to an opaque viscous liquid.
All 3 corneas treated with Bis(dimethylamino)methylvinylsilane showed complete opacity of the tissue. All 3 corneas were covered with a thin whitish layer which could not be removed by routine non-invasive methods, such as repeated washing.
The following mean in vitro irritation score was calculated:
319.83
Therefore the test item was classified into UN GHS Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)