Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
Sept., 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX, UK, protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", dated February 994
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: test system: freshly isolated bovine cornea
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Rationale: Recommended by INVITTOX, UK, protocol no. 98

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: 0.9% (w/v) NaCl in deionised water (saline; negative control), 2-Ethoxyethanol (positive control)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Duration of treatment / exposure:
10 min
Observation period (in vivo):
Third opacity reading (t130) done 2h after 2nd opacity measurement (t10). The optical density was measured after additional 90 min.
Number of animals or in vitro replicates:
3 replicate corneas for each treatment group (negative control solution, test item and positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): test item or control items, respectively, were rinsed off from the application side with saline
- Time after start of exposure: 10 min

SCORING SYSTEM: This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of the irritant effect of the test item.

TOOL USED TO ASSESS SCORE: For the measurement of the corneae opacity the OP_KiT opacitometer (Electro Design, 63-Riom France) was used. Permeability of the cornea possibly caused by the test item, was determined with 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution). The optical density was measured spectrophotometrically at 490 nm (OD490).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
cornea #1 and #2 (test item)
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corrected value; IVIS ≤ 3 -> No Category (UN GHS, Eye damage)
Irritation parameter:
in vitro irritation score
Run / experiment:
cornea #3 (test item)
Value:
1.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corrected value; IVIS ≤ 3 -> No Category (UN GHS, Eye damage)
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (test item)
Value:
0.79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: IVIS ≤ 3 -> No Category (UN GHS, Eye damage)
Other effects / acceptance of results:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro score was calculated as 0.47.
The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as severe eye irritant. The mean in vitro score was calculated as 70.31.

Any other information on results incl. tables

Results after 10 Minutes Incubation Time

Test Group      Opacity value = Difference (t130 -t0) of Opacity     Permeability at 490 nm (OD490)     IVIS    Proposed in vitro irritation scale    
   Mean    Mean    Mean
 Negative Control        0  -0.33        0.054  0.054        0.81  0.47        Non eye irritant      
 -1  0.052  -0.22
 0  0.055  0.83
 Positive Control        53.33*     0.740*     64.44  70.31        Severe eye irritant      
 52.33*     0.802*     64.37
 73.33*     0.585*     82.11
 Test Item        0.33*     0.015*     0.56  0.79        Not corrosive / irritant(CLP/GHS)      
 0.33*     0.001*     0.35
 1.33*     0.008*     1.46

*corrected values

The validity criteria and findings are given in the following table:

 Parameter  Criterion  Found  Assessment
 IVIS of negative control  ≤ 3  0.47  valid
 IVIS of positive control  59.17 - 108.11  70.31  valid

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study and under the experimental conditions reported, according to the CLP/GHS classification the test item is not considered to be corrosive/irritant to the eye. The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item. This single in vitro test can serve as a stand-alone test as it was consistently shown that the test item does not require classification (eye damage).
Executive summary:

This in vitro GLP study according to OECD Guideline 437 was performed to assess the corneal irritation and damage potential of the test substance by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t10). Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was measured a third time (t130).

After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 1 °C in a horizontal position. The liquid coming out was measured spectrophotometrically.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as severe eye irritant.

The test item did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was 0.79.

In conclusion, it can be stated that in this study and under the experimental conditions reported, according to the CLP/GHS classification the test item is not considered to be corrosive/irritant to the eye.