N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 437), performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

as of September, 2009

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

235-426-8

EC Name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

12225-08-0

Molecular formula:

C27H24N6O7S

IUPAC Name:

4-{[2,5-dimethoxy-4-(methylsulfamoyl)phenyl]diazenyl}-3-hydroxy-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Test system

Amount / concentration applied:

A 20% (w/v) suspension of the test item in physiological saline was prepared. Each 0.75 mL of the suspension were applied to three bovine corneas.

Duration of treatment / exposure:

The corneas were exposed to the test item suspension for 240 minutes.

Details on study design:

After a first opacity measurement of the fresh bovine corneae (t0), 0.75 mL per cornea of a 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium =cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).
After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically (OD 490) the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 2.46

Other effects / acceptance of results:

Relative to the negative control, the test item did not cause any increase of the corneal permeability, and only very slight opacity occurred. The calculated mean in vitro irritation score of 2.46 was below the threshold of = 55.1. Therefore the test item is not corrosive / not severe irritant to the eye according to OECD 437. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity of the corneae corresponding to a corrosive / severe irritant to the eye.

Any other information on results incl. tables

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	1	1.00	0.049	0.057	1.73	1.86	Non corrosive / not severe irritant
	1		0.079		2.19
	1		0.044		1.66
Positive Control	174.00*		0.009*		174.13	183.48	Corrosive / severe irritant
	175.00*		0.028*		175.42
	201.00*		- 0.007*		200.89
Test item	3.00*		0.014*		3.21	2.46	Non corrosive / not severe irritant
	2.00*		- 0.001*		1.98
	2.00*		0.014*		2.21

*corrected values

Applicant's summary and conclusion

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not corrosive / not severely irritating to the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneas (OECD 437: bovine corneal opacity and permeability assay; GLP).

Prior to the application, opacity of the fresh bovine corneas was measured (t₀), and 600.91 mg of the test item were suspended in 3.0 mL saline (20% (w/v)) using ultrasonic technique for five minutes. Since the test item could not be suspended homogeneously, each 0.75 mL of the so prepared suspension was distributed to each cornea. Thereby it was taken care of that the corneas were evenly covered with the test item. The positive control was 10% (w/v) Benzalkonium chloride in saline. Saline was used as negative control item.

The exposed corneas were incubated for 240 minutes at 32 +/- 1 °C in complete medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 +/- 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneas could be observed (mean in vitro irritation score 1.86).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity effects (mean in vitro irritation score 183.48) corresponding to a corrosive /severe irritant to the eye.

Relative to the negative control, the test item did not cause any increase of the corneal permeability, and only very slight opacity occurred. The calculated mean in vitro irritation score of 2.46 was below the threshold of >= 55.1. Therefore the test item is not corrosive / not severe irritant to the eye according to OECD 437.