Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-13 to 2018-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 09 October 2017)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,​2'-​[1,​2-​ethanediylbis(oxy)​]​bis-​, reaction products with 3-​(triethoxysilyl)​-​1-​propanamine

EC Number:

813-152-5

Cas Number:

152261-44-4

Molecular formula:

Unspecified

IUPAC Name:

Ethanol, 2,​2'-​[1,​2-​ethanediylbis(oxy)​]​bis-​, reaction products with 3-​(triethoxysilyl)​-​1-​propanamine

Test material form:

liquid

Test animals / tissue source

Species:

other: bovine cornea

Strain:

not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 μL

Duration of treatment / exposure:

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).

Duration of post- treatment incubation (in vitro):

2 h before illuminance measurement followed by 90 min before determination of optical density

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%

Details on study design:

Preparation of the Corneas
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, 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.
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 (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). 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) was switched on 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout should lie 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 should display values between 300-310 lux and between 95-105 lux. If this is not the case, the calibration procedure has to be repeated. The calibration procedure is performed before each 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. 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 test substance or the control substance was introduced into the anterior chamber. 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 (without phenol red). The anterior chamber was refilled with complete RPMI 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. 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).



Test Groups
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%
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 (=I zero) 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 reevaluated 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 below:
Evaluation of the BCOP Assay
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No 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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

test item

Value:

46.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.81

Positive controls validity:

valid

Remarks:

63.38

Other effects / acceptance of results:

No prediction can be made regarding the classification of the test substance according to the evaluation criteria.

Applicant's summary and conclusion

Interpretation of results:

other: An IVIS of 46.32 was determined which is close to the cut-off for classification as severely eye damaging of 55.1. Due to the severe corneal effects observed, the test substance is classified as severely damaging to eyes.

Conclusions:

No prediction can be made regarding the classification of the test substance Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine according to the evaluation criteria.

Executive summary:

Summary Results

The eye irritancy potential of Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item:  tested as provided by the sponsor

Visual Observation after treatment:  All 3 corneas treated with Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine showed opacity of the tissue.

Mean in vitro irritation score:                    46.32

 

		UN GHS No Category
x		No prediction can be made
		UN GHS Category 1

Classification                                              

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

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.

Conclusion

No prediction can be made regarding the classification of the test substance Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine according to the evaluation criteria.