Coupling reaction products of diazotized 2-amino-6-[(2-substitued ethyl)sulfonyl]naphthalene-1-substituted acid with 4-({4-chloro-6-[ethyl(3-{[2-(substituted oxy)ethyl]sulfonyl}phenyl)amino]-heteromonocycl-2-yl}amino)-5-hydroxynaphthalene-2,7-disubstituted acid and their sodium and potassium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay"

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- public name of test material : Coupling reaction on diazotized (aminonaphthalen)sulfonyl)ethanol polysulfanate with 6-chloro-N-ethyl-N-(3-(ethylsulfonyl)phenyl)-N'-naphthalen-1-yl-1,3,5-triazine polysulfonate, polyhydroxide, polyamine, sodium and potassium salts
- Substance type: textile dyestuff
- Physical state: red powder
- Analytical purity: ca. 63 % of all colored components
- Lot/batch No.: BOP 07-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C

Test animals / tissue source

Species:

other: in vitro study with bovine cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes were collected from the abattoir and transported in Hank’s BSS supplemented with streptomycin / penicillin at room temperature. The corneae were isolated immediately after delivery of the eyes to the laboratory.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects and discarded when indicated. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects. Each cornea was mounted in a cornea holder; subsequently, both compartments of the holder were filled with complete medium and allowed to equilibrate for about 1 hour at 32 ± 2 °C in a water-bath.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The positive control was 10% (w/v) Benzalconium chloride. 0.9% (w/v) saline was used as negative control item.

Amount / concentration applied:

ca 100 mg test item

Duration of treatment / exposure:

240 min (+/-5 min)

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

Number of Corneae per Group: 3
Number of Test Item Group: 1
Number of Negative Control Group: 1
Number of Positive Control Group: 1
Total Number of Corneae: 9

Details on study design:

Study design
The corneae were distributed as follows:
Negative Control: 3
Positive Control: 3
Test Item: 3
Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item (approx. 100 mg) or negative or positive control at a volume of 1.0 mL each on the surface of the corneae and was incubated at 32 ± 2 °C in the water-bath in a vertical position.
The positive control was 10% (w/v) Benzalconium chloride. 0.9% (w/v) saline was used as negative control item.
The incubation time lasted 240 minutes (± 5 minutes).
After the test item or control items, respectively, were rinsed off from the application side by changing cMEM several times, fresh cMEM was replaced in both compartments and opacity was measured (t240).
In the second step of the assay, permeability of the cornea possibly caused by the test item, was determined. Fresh complete medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5 % dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for further 90 minutes at 32 ± 2 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After recording the basal opacity of all corneae, the values of all corneae were noted. Sets of three corneae were used for treatment with the test item and the negative and positive controls.
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was plugged. The cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a water-bath at 32 ± 2 °C for 240 minutes. Afterwards, the opacity was measured again (t240).

Permeability Determination
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) fluorescein solution. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 2 °C. Complete medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

Results and discussion

In vivo

Irritant / corrosive response data:

With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro score was calculated as 2.59.

The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The mean in vitro score was calculated as 221.84.

The test material did not cause any permeability of the corneae compared with the results of the negative control. Due to the dying property of the test item the corneae were coloured but still translucent after the treatment period, thus the increase of the opacity value compared with the results of the negative control is most probably caused by the dying effect. The calculated mean in vitro score was 48.90 and therefore, the test item has to be classified as moderate eye irritant according to INVITTOX protocol no. 98, but the actual effect of the present test might be weaker than the irritation score indicates.

Any other information on results incl. tables

Results after 240 Minutes Incubation Time

Test group	Opacity value		Permeability		In vitro score	Mean in vitro score	Proposed in vitro irritation scale
		Mean		Mean
Negative control	0	0.33	0.116	0.150	1.74	2.59	Non eye irritant
	1		0.147		3.20
	0		0.188		2.82
Positive control	244.7 *	213.7	0.546 *	0.545	252.86	221.84	Very severe eye irritant
	191.7 *		0.603 *		200.72
	204.7 *		0.486 *		211.95
test material	44.7 *	48.7	-0.006 *	0.015	44.58	48.90	Moderate eye irritant
	54.7 *		0.040 *		55.27
	46.7 *		0.011 *		46.83

* corrected values

Applicant's summary and conclusion

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: INVITTOX protocol no. 98

Conclusions:

In this study and under the experimental conditions reported, the test material is considered to be a moderate eye irritant according to the criteria proposed in the INVITTOX protocol no. 98 and causes no irreversible effects to the eyes.
Based upon the classification criteria according to „Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008“ the test item is not classified as Eye Effects Category 1 (irreversible effects, eye corrosion). Prediction of eye irritation is not possible using this model.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of test material by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437 and INVITTOX protocol no. 98.

After a first opacity measurement of the fresh bovine corneae (t0), the test material, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 2 °C in cMEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium). 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 while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The coming out liquid was measured spectrophotometrically.

With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant.

The test material did not cause any permeability of the corneae compared with the results of the negative control. Due to the dying property of the test item the corneae were coloured but still translucent after the treatment period, thus the increase of the opacity value compared with the results of the negative control is most probably caused by the dying effect. The calculated mean in vitro score was 48.90 and therefore, the test item has to be classified as moderate eye irritant. But the actual effect of the present test might be weaker than the irritation score indicates.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test material is considered to be a moderate eye irritant according to the criteria proposed in the INVITTOX protocol no. 98.

Based upon the classification criteria according to „Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008“ the test item is not classified as Eye Effects Category 1 (irreversible effects, eye corrosion). Prediction of eye irritation is not possible using this model.