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Toxicological information

Eye irritation

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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

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.