Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-02-28
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: The Bovine Corneal Opacity and Permeability Assay (BCOP) according to IVITTOX (UK) protocol no. 98
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd. UK, Prodceure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40840/A TE
- Substance type: colouring dye
- Physical state: solid, dark red powder
- Analytical purity: approx. 78.9% org. part (NA-salt), MC: 37.7%, Oligomers: 19.2%
- Lot/batch No.: ROE 358 BOP 01/07
- Expiration date of the lot/batch: June 30, 2012
- Storage condition of test material: At room temperature at about 20°C

Test animals / tissue source

Species:
other: no animals, in vitro test
Details on test animals or tissues and environmental conditions:
not applicable

Test system

Vehicle:
other: in vitro test
Amount / concentration applied:
TEST MATERIAL
- Amount applied (volume or weight with unit): 160 mg
Duration of treatment / exposure:
240 min.
Number of animals or in vitro replicates:
no animals
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

Any other information on results incl. tables

Results after 240 mi. 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     0.044    2.66    
  Negative control  2  1.67  0.046  0.045  2.69  2.35  Non eye irritant
  Negative control  1    0.046    1.69    
  Positive Control

 111.33   

 2.0297   

 141.78    
  Positive Control

 117.33   

 2.0307   

 147.79 148.72   Severe eye irritant
  Positive Control

 126.33   

 2.0167   

 156.58    
  FAT 40840/A TE

 11.33   

 0.0267   

 11.73    
  FAT 40840/A TE

 11.33   

 0.0127   

 11.52  11.70  Mild eye irritant
  FAT 40840/A TE

 11.33   

 0.0337   

 11.84    

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 FAT 40840/A TE caused opacity and permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 11.70 and therefore, the test item was classified as mild eye irritant.

Applicant's summary and conclusion

Interpretation of results:
other: mild 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 FAT 40840/A TE is considered to be a mild eye irritant.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of FAT 40840/A TE by means of the BCOP assay using fresh bovine corneas. 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. 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 FAT 40840/A TE caused opacity and permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 11.70 and therefore, the test item was classified as mild eye irritant. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item FAT 40840/A TE is considered to be a mild eye irritant.