N,N-dibutylbutan-1-aminium bis[2-(hydroxy-kO)benzoato(2-)-kO]borate(1-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-16 May 2013 (in-life dates)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline (OECD 437) compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

EC Number:

700-906-2

Cas Number:

22450-96-0

Molecular formula:

C26H36BNO6

IUPAC Name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

Constituent 2

Reference substance name:

SABoTBA

IUPAC Name:

SABoTBA

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SABoTBA
- Purity: 98.77%
- Purity test date: 08 April 2014
- Lot/batch No.: 12342
- Expiration date of the lot/batch:Stable under recommended storage conditions
- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes, supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (excised at 12.40 hours, 14 May 2013). Instructions were given to avoid damaging the corneas during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle. The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 14.41 hours, 14 May 2013).

Test system

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

750 µL of a 20% w/w solution in 0.9% sodium chloride

Duration of treatment / exposure:

4h (± 5 min)

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

Preparation of corneas
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1% penicillin/streptomycin solution until all the corneas had been dissected. Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% penicillin/streptomycin solution prior to mounting.
The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. Each cornea was gently flattened over the O-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with HBSS plus 1% penicillin/streptomycin, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders. The holders were then plugged and incubated, in an upright position, overnight at room temperature (approximately 20°C).
After overnight incubation at room temperature (approximately 20°C) the HBSS plus 1% penicillin/streptomycin was removed from both chambers. Both chambers were filled with cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. The corneas were then incubated in the upright position for 60 minutes ± 5 minutes at 32°C± 1°C in a waterbath. The waterbath temperature remained within the limits of 32°C ± 1°C throughout the experiment.
At the end of the 60 minute incubation period, the medium was removed from both the anterior and posterior compartments using a pipette tip attached to a vacuum pump. The compartments were refilled with fresh cMEM (posterior chamber first). Again care was taken to ensure no air bubbles were present within the holders. The posterior compartment was then plugged and the basal opacity measurements performed.

Opacity measurement
The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.

Treatment groups
Corneas were treated in triplicate with either the test substance, positive control (imidazole) or negative control (0.9% sodium chloride solution).

Treatment of corneas
The test substance, SABoTBA, a white crystalline powder, and the positive control material (imidazole) were tested at 20% (w/w) in 0.9% sodium chloride solution. The test substance was mixed during preparation using a mortar and pestle. The 0.9% sodium chloride solution was added to achieve the desired concentration.
Immediately prior to treatment, the medium was removed from the anterior compartment of the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all excess liquid had been removed. Seven hundred and fifty µL (750 µL) of test substance, negative control (0.9% sodium chloride solution) or positive control material (imidazole 20% w/w solution) was introduced in to the anterior part of the holder. The test substance at 20% w/w formed a white suspension and was added to the anterior compartment using a 1 mL syringe via the top port. Following application the anterior compartment was plugged. The holder was slightly rotated to ensure uniform distribution of the test substance over the surface of the cornea. Each holder was incubated in a horizontal position at 32°C ± 1°C for 4 hours ± 5 minutes in a waterbath.
Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed, at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. The corneas were gently rinsed with the wash medium until the medium was clear and the colour unchanged. The corneas treated with the test substance required 7 washes. Following completion of the wash step, the medium was removed from both compartments and re-filled with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.
Throughout the assay the corneas were examined for opaque spots or other irregularities.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Summary of results

Sample	Opacity ± SD	Permeability ± SD	In vitro irritancy Score ± SD	In vitro classification
SABoTBA	1.000 ± 1.000	0.025 ± 0.038	1.4 ± 1.5	Non-corrosive/Non-severe irritant
Imidazole	126.333 ± 24.583	3.212 ± 0.563	174.5 ± 17.6	Corrosive/Severe irritant
0.9% saline	0.000 ± 0.000	0.078 ± 0.004	NA	NA

   

Assay validity

The positive control, imidazole,elicited an In Vitro Irritancy Score of 174.5. This value was within the historical range (mean ± 2 x SD 126.0 – 185.4) for the assays performed to date. 

 The negative control, 0.9% saline, opacity mean change value was 0.000 which was below the maximum acceptance value of 3.0. The permeability mean of the negative control was 0.078 which was below the maximum acceptance value of 0.1.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance, SABoTBA, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was predicted to be a non-corrosive/non-severe eye irritant.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance, SABoTBA. Imidazole was tested in parallel as a positive control.

The assay used isolated bovine corneas to assess the ocular corrosivity or severe irritancy potential of the test substance in vitro. The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which was used to classify the test substance as potential eye irritants according to OECD Guideline 437.

The test substance,SABoTBA, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was predicted to be a non-corrosive/non-severe eye irritant.