[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-12

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012

Test animals / tissue source

Species:

other: no animals, in vitro test

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable, in vitro test

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 0.1g

VEHICLE
no vehicle

Duration of treatment / exposure:

240 min (± 5 min)

Observation period (in vivo):

not applicable, in vitro test

Number of animals or in vitro replicates:

not applicable, in vitro test

Details on study design:

Test System: freshly isolated bovine corneas
Number of Conreas per Group: 3
Number of Test Item Group: 1
Number of Negative Control Group: 1
Number of Positive Control Group: 1
Total number of Corneas: 9

After a first opacity measurement of the fresh bovine corneas (t0), the test item FAT 40840/A
TE, the positive, and the negative controls were applied to corneas and incubated for 240
minutes at 32 ± 2 °C in cMEM medium, supplemented with 10% FCS. 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 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.

Results and discussion

In vivo

Results

Irritation parameter:

other: In Vitro Test. See Point "Remarks on Results"

Basis:

other: 3 corneas

Time point:

other: after 240min incubation time

Score:

> 42 - < 151

Max. score:

151

Reversibility:

other: not applicable

Remarks on result:

other: In Vitro Score

Any other information on results incl. tables

Results after 240 min. Incubation time

Test Group	Opacity value = Difference (t240 -t0) of Opacity		Permeability at 490 nm (OD490)*		In vitro Score	Mean in vitro Score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative control	2		0.0576		2.86
Negative control	1	1.33	0.0512	0.056	1.77	2.17	Non eye irritant
Negative control	1		0.0594		1.89
Positive Control	92.67		2.3085		127.29
Positive Control	137.67		2.2400		171.27	164.27	very severe eye irritant
Positive Control	159.67		2.3058		194.25
FAT 40841/A TE	41.67		0.0267		42.09
FAT 40841/A TE	88.67		0.0127		88.71	93.86	very severe eye irritant
FAT 40841/A TE	147.67		0.2076		150.78

In Vitro Score

In vitro score	Proposed in vitro Scale
0 -3	non eye irritant
3.1 - 25	mild eye irritant
25.1 - 55	moderate eye irritant
55.1 - 80	severe eye irritant
>80.1	very severe eye irritant

With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneas could be observed. The positive control (10% (v/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneas and therefore, is classified as very severe eye irritant.

The test item test material caused opacity and permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 93.86 and therefore, the test item was classified as very severe eye irritant. The test item caused coloring of the cornea which could not be rinsed off even after stringent washing. Therefore the positive effect may be caused by the coloring effect.

Applicant's summary and conclusion

Interpretation of results:

other: very severe eye irritant

Remarks:

Criteria used for interpretation of results: other: INVITOX (UK) protocol No. 98

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions
reported, the test item test material 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 test material by means of the BCOP assay using fresh bovine corneas.

After a first opacity measurement of the fresh bovine corneas (t0), the neat test item test material, the positive, and the negative controls were applied to corneas and incubated for 240 minutes at 32 ± 2 °C in cMEM medium, supplemented with 10% FCS. 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 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 corneas could be observed.

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

The test item test material caused opacity and permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 93.86 and therefore, the test item was classified as very severe eye irritant. The test item caused coloring of the cornea which could not be rinsed off even after stringent washing. Therefore the positive effect may be caused by the coloring effect.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item test material is considered to be a very severe eye irritant.