Alkane-alpha,omega-diyl bis{[(triethoxysilyl)propyl]carbamate}_E2

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-23 to 2015-02-12

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted July 26, 2013

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

EC Guidelines Council Regulation (EC) No. 1152/2010 method B.47 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants“ (published in the Official Journal of the European Union L324, December 09, 2010

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Reaction mass of 2,2,4-trimethylhexane-1,6-diylbis{[3-(triethoxysilyl)propyl]carbamate} and 2,4,4-trimethylhexane-1,6-diylbis{[3-(triethoxysilyl)propyl]carbamate} manufactured by Evonik Degussa GmbH. Batch No. IPES-Addukt 7W of 2014-06-23, purity: 95.05.%.

Test animals / tissue source

Species:

other: Bovine eyes from cattle in the age range of 6 to 12 months

Details on test animals or tissues and environmental conditions:

Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse1. To minimize deterioration and bacterial
contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution2 (HBSS) containing 1% Penicillin/Streptomycin3.
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation.
Only corneas from eyes free of defects were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive control item: 1% NaOH solution in aqua ad iniectabilia, Solvent control item: 0.9% sodium chloride solution

Amount / concentration applied:

750 µl test item was used as supplied.
750 µl solvent control item, 0.9% sodium chloride solution
750 µl positive control item, 1% NaOH solution in aqua ad iniectabilia

Duration of treatment / exposure:

Exposure period: 10 minutes

Observation period (in vivo):

After rinsing the corneas were incubated at 32°C ± 1°C for two hours. After this post incubation period, the corneas were examined.

Number of animals or in vitro replicates:

Three corneas were used for each treatment group (test item, solvent control and positive control).

Details on study design:

PREPARATION OF BOVINE EYES
- Corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium)
chambers
- The chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM)
- Corneal holder was equilibrated at 32±1°C for at least one hour
- After equilibration period, fresh pre-warmed EMEM was added to both chambers
- Baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation,
neovascularisation) or an opacity >7 opacity units were discarded
- Mean opacity of all equilibrated corneas was calculated by use of an opacitometer
- A minimum of three corneas with opacity values close to the median value for all corneas were selected as solvent control corneas
- The remaining corneas were then distributed into treatment, negative and positive control groups
ADMINISTRATION
- Three corneas were used for each treatment group
Solvent control item: 0.9% sodium chloride solution
Positive control item: 1% NaOH solution in aqua ad iniectabilia
Test item: test item was used as supplied
- Exposure period: 10 minutes
- After exposure period of 10 minutes the test item, solvent control and positive control, were removed from each chamber and epithelium was
washed with EMEM at least three times
- As the test item was highly viscous and not soluble in EMEM, the epithelium was washed additionally with 50% dimethylsulfoxide (DMSO) in aqua
ad iniectabilia
- Subsequently, the epithelium was washed with EMEM containing phenol red at least three times in order to detect pH-changes of the medium
caused by residues of the test item or control items
- Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible
- Corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber
- Chamber was then filled with EMEM without phenol red
- After rinsing the corneas were incubated at 32°C ± 1°C for two hours. After this post incubation period, the corneas were examined
EXAMINATION
- Corneal injury was assessed by evaluating the opacity and permeability of the cornea
- Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer
resulting in opacity values measured on a continuous scale
- To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior
chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM
- The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes
- Amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader
(Tecan Sunrise Magellan Version 6.412).
- Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values
based upon a visible light spectrophotometer using a standard 1 cm path length

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

BCOP test

Value:

< 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Conclusions:

Under the present test conditions the test item, tested in the in vitro BCOP test method, had an IVIS value of -0.362, which is below the cut-off value
of 3 (UN GHS no category). Test item does neither induce serious eye damage nor serious eye irritation in this in vitro test system. Therefore, no
classification concerning hazard class “serious eye damage/eye irritation” is necessary.

Executive summary:

The purpose of this study was to determinea possible potency of the test item of being 'ocular corrosive and severe irritant' employing an in vitro system.The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro.In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability inisolated corneas from bovine eyes.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber.The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, solvent control and positive control). The liquid test item was used undiluted as recommended in the test guideline 437.0.9% NaCl solution was used as the solvent control and 1% NaOH in aqua ad iniectabilia as the positive control item.

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was10 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The acceptance criteria of validity were fulfilled in this test.

Following treatment with test item a meanopacity and a mean permeability value of <0.01 compared to the solvent control were

determined. An IVIS of -0.362 was calculated. The calculated IVIS was below the cut-off value of 3 (UN GHS no category). Hence, the test item does neither induce serious eye damage nor serious eye irritation in this in vitro test system. Therefore, no classification concerning hazard class “serious eye damage/eye irritation” is necessary.

 

 

Conclusion

Under the present test conditions test item, tested in the in vitro BCOP test method, had an IVIS value of-0.362,which is below the cut-off value of 3 (UN GHS no category).Test item does neither induce serious eye damage nor serious eye irritation in this in vitro test system. Therefore, no classification concerning hazard class “serious eye damage/eye irritation” is necessary.