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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation
Remarks:
other: in vitro study but an accepted alternative to in vivo testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guideline 437 and in compliance with principles of Good Laboratory Practices
Cross-reference
Reason / purpose:
reference to other 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:
other: OECD guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September, 2009).
Qualifier:
according to
Guideline:
other: The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed according to the INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", dated February 1994.
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Principles of method if other than guideline:
-
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: 1,1-dichloroethylene
Batch-No.: 0B 135
Production Date: February 05, 2010
Purity: > 99%
Storage: At room temperature, protected from light, in N2 atmosphere,original container
Expiration Date: February 04, 2011

Test animals / tissue source

Species:
other: in vitro test: freshly isolated bovine cornea
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea
Rationale: Recommended by INVITTOX, UK, INVITTOX protocol No. 98
Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany

Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above. Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 2 °C in a water-bath. At the end of the incubation period, the complete medium was removed from both compartments and replaced with fresh cMEM, and the basal opacity was determined (t0). For measurement, the posterior compartment was plugged and the anterior compartment was unplugged.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: negative control (0.9% (w/v) NaCl (physiological salt solution, produced in-house)) and positive control (2-Ethoxyethanol Lot Number: 0001430432)
Amount / concentration applied:
Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae and was incubated at 32 ± 2 °C in the water-bath, while the corneae were in a horizontal position.
Duration of treatment / exposure:
The incubation time lasted ten minutes (± 30 seconds)
Observation period (in vivo):
After the test item or control items were rinsed off from the application side by changing cMEM several times, in minimum three times, fresh cMEM was added and opacity was measured (t10). The corneae were then incubated at 32 ± 2 °C for further two hours in a vertical position, followed by a third opacity reading (t130). In the second step of the assay, permeability of the cornea possibly caused by the test item, was determined. Fresh complete medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 2 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490). For the measurement of the corneae opacity the OP_KiT opacitometer (Electro Design, 63-Riom France) was used.
Number of animals or in vitro replicates:
Groups Number of Corneae
Negative Control 3
Positive Control 3
Test Item 3
Details on study design:
EVALUATION OF RESULTS

Opacity
The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

In Vitro Score Calculation
The following formula was used to determine the in vitro score of the negative control:
In vitro Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro score of the positive control and the test item:
In vitro Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro score was calculated for each individual treatment and positive control cornea. The mean in vitro score value of each treated group was calculated from the individual in vitro score values.
Depending on the score obtained, the test item was classified into one of the following
categories:
In vitro Score Proposed in vitro irritation 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

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: in vitro score
Basis:
mean
Remarks:
Negative control
Time point:
other: 10 +120 min.
Score:
0.82
Remarks on result:
other: Non eye irritant
Irritation parameter:
other: in vitro score
Basis:
mean
Remarks:
Positive control
Time point:
other: 10 +120 min.
Score:
79.2
Remarks on result:
other: Severe eye irritant
Irritation parameter:
other: in vitro score
Basis:
mean
Remarks:
1,1-dichloroethene
Time point:
other: 10 +120 min.
Score:
43.9
Remarks on result:
other: moderate eye irritant
Irritant / corrosive response data:
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 0.82. 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 79.20. The test item 1,1-dichloroethylene caused only a slight increase of opacity values but a distinct increase of the permeability values of the corneae compared with the results of the negative control. The calculated mean in vitro score was 43.90 and therefore, the test item was classified as moderate eye irritant.

Any other information on results incl. tables

Results

 Test Group   Opacity value = Difference (t130-t0) of Opacity   Permeability at 490 nm (OD490)*   In vitro Score   Mean in vitroScore   Proposed in vitroIrritation Scale 
     Mean     Mean       
 Negative Control   - 1   0.00   0.052   0.055   - 0.22   0.82   Non eye irritant 
 Negative Control   0   0.051   0.77 
 Negative Control   1   0.061   1.92 
 Positive Control   66.00   0.640   75.61   79.20   Severe eye irritant 
 Positive Control   68.00   0.874   81.12 
 Positive Control   69.00   0.791   80.87 
1,1-dichloroethylene   10.00   2.312   44.69   43.90   Moderate eye irritant 
1,1-dichloroethylene   9.00   2.306   43.60 
1,1-dichloroethylene  8.00   2.360   43.41 

* corrected values

Applicant's summary and conclusion

Interpretation of results:
moderately irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions reported, the test item 1,1-dichloroethylene is considered to be a moderate eye irritant.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of 1,1-dichloroethylene 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 1,1-dichloroethylene, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 2 °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 ± 2 °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 ± 2 °C in a horizontal position. The liquid coming out 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 positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as severe eye irritant. The test item 1,1-dichloroethylene caused only a slight increase of opacity values but a distinct increase of the permeability values of the corneae compared with the results of the negative control. The calculated mean in vitro score was 43.90 and therefore, the test item was classified as moderate eye irritant.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item 1,1-dichloroethylene is considered to be a moderate eye irritant. Therefore the test item needs not be classified as R41 or GHS category 1, but requires a classification as R36 or GHS category 2.