Bis(dimethylamino)methylvinylsilane

Eye irritation

Administrative data

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

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 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)

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

Test animals / tissue source

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.

Test system

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).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

319.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.