2-[(8-amino-7-{[4-substituted-2-sulfonatophenyl]diazenyl}-1-hydroxy-3,6-disulfonaphthalen-2-yl)diazenyl]-4-[(4-chloro-6-{[3-(substituted)phenyl](ethyl)amino}-heteromonocycl-2-yl)amino]arylsulfonic acid, potassium and sodium 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 for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September, 2009).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The Bovine Corneal Opacity and Permeability Assay (BCOP) will be performed according to the INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", dated February 1994. �

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): FAT 40849/A TE
- Substance type: reactive dyestuff for cellulose fibers
- Physical state: blue powder
- Analytical purity: 76.9% of all colored components
- Lot/batch No.: TZ 5463 / BOP 03-09
- Expiration date of the lot/batch: 2014-06-30
- Storage condition of test material: At room temperature at about 20 °C

Test animals / tissue source

Species:

other: in vitro study

Strain:

other: in vitro study

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro study

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:

not applicable

Details on study design:

This test is designed to measure the opacity of the cornea by quantifying the ability of light of pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein solution. Comparison of the opacity before and after exposure to test item and determination of permeability after treatment provide an indication of the irritant effect of the test item.
For this purpose the inflamed in vitro changes of the bovine cornea after application of the test item will be measured with regard to the opacity and permeability of dye. The results of both criteria will be combined. The resulting in vitro irritation factor will be compared with a criterion scale, produced prior to the experiment. The changes are classified into four grades:
mild, moderate, severe, very severe.

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro study. see item "any other information on results

Basis:

other: 3 corneas

Time point:

other: after 240 min treatment

Score:

ca. 458

Max. score:

465

Reversibility:

other: not applicable

Remarks on result:

other: in vitro study

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 vitroScore	Meanin vitroScore	Proposedin vitroIrritation Scale
		Mean		Mean
Negative Control	0	0.33	0.116	0.150	1.74	2.59	Nonan
Negative Control	1		0.147		3.20
Negative Control	0		0.188		2.82
Positive Control	244.67*	213.67	0.546*		252.86	221.84	very severe
Positive Control	191.67*		0.603*		200.72
Positive Control	204.67*		0.486*		211.95
FAT 40842/A TE	427.67*	427.67	2.498*	2.019	465.13	457.95	very severe
FAT 40842/A TE	427.67*		1.986*		457.45
FAT 40842/A TE	427.67*		1.573*		451.26

*corrected values

Positive Control

10% (w/v) Benzalconium chloride in 0.9% (w/v) NaCl (physiological salt solution, produced in-house) served as positive control.

Negative Control

0.9% (w/v) NaCl (physiological salt solution, produced in-house) served as negative control.

SCORING

In vitroScore	Proposedin vitroirritation Scale
0 - 3	Non eye irritant
3.1 – 25	Mild eye irritant
25.1 – 55	Moderate eye irritant
55.1 – 80	Severe eye irritant
> 80	Very severe eye irritant

Applicant's summary and conclusion

Interpretation of results:

highly irritating

Remarks:

Migrated information under the experimental conditions reported, the test item FAT 40849/A TE is considered to be a very severe eye irritant. Criteria used for interpretation of results: other: Criteria for Determination of a Valid Test The test was acceptable if the positive control caused an at least moderate effect.

Conclusions:

In this study and under the experimental conditions
reported, the test item FAT 40849/A TE is considered to be a very severe eye irritant.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of FAT 40849/A TE by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), each about 100 mg of the test item FAT 40849/A TE, 1 mL of the positive control, and also 1 mL of the negative control were applied to the corneae and incubated for 240 minutes at 32 ± 2 °C in complete medium (cMEM medium supplemented with sodium bicarbonate and Lglutamine and 1% fetal calf serum (FCS)). 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 about 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 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 item FAT 40849/A TE caused clear opacity and permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was 457.95 and therefore, the test item was classified as very severe eye irritant.