Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 12 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identification: MEDOL-10
Chemical Name: (2-Ethyl-2-methyl-1,3-dioxolan-4-yl)methyl acrylate
CAS No.: 69701-99-1
Batch: 09892701
Purity: 99.9%
Appearance: Colourless liquid
Expiry Date: 06 March 2018
Storage Conditions: At room temperature, protected from light
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Collection of Bovine Eyes Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 Minutes
Number of animals or in vitro replicates:
3 corneas per treatment
Details on study design:
Experimental Design and Study Conduct

Preparation of Corneae
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.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. 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 incubation 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 about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Exposure of the Corneae to the Test Groups
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3

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. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes. After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). The opacity measurement is described below.
In the second step of the assay, permeability of the corneae was determined. The permeability measurement is described below.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer. The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item, negative, and positive controls.

Data Evaluation
Opacity The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0). The average change in opacity of the negative control corneae is calculated and this value is 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 is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Criteria for Determination of a Valid Test
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
> 0.63 - < 2.9
Negative controls validity:
valid
Remarks:
1.23
Positive controls validity:
valid
Remarks:
76.41
Remarks on result:
no indication of irritation

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

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0

0.078

0.082

1.17

1.23

Not categorized

0

0.082

1.23

0

0.085

1.28

Positive Control

65.00*

0.794*

76.92

76.41

Category 1

70.00*

0.696*

80.45

62.00*

0.658*

71.88

MEDOL-10

3.00*

-0.007*

2.90

2.93

Not categorized

3.00*

-0.029*

2.57

1.00*

-0.025*

0.63

* corrected values

Applicant's summary and conclusion

Interpretation of results:
other: Non-irritating, not requiring classification
Remarks:
Criteria used for interpretation of results: EU CLP and UN GHS.
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, MEDOL-10 is not categorized (GHS).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of MEDOL-10 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 fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.  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 corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).  

Relative to the negative control, the test item MEDOL-10 did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 2.03. According to OECD 437 the test item is not categorized (GHS).