Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 13, 2017 - January 10, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL (150 mg/750 µL)
- Concentration: The test item was prepared as a 20% (w/v) suspension in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

VEHICLE
- Amount(s) applied: 750 µL
- Concentration: 0.9% sodium chloride solution
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three corneas were used per group (negative control, positive control or test item group)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprém, Hungary) 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 without stretching the cornea. Afterwards, 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 form.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g.scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES
Three corneas were used per group (negative control, positive control or test item group). Therefore, a total number of 9 corneas was used in this study.

NEGATIVE CONTROL USED
Negative/Vehicle Control: 0.9% sodium chloride solution, B. Braun Melsungen AG, Batch: 16455011,

POSITIVE CONTROL USED
Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v). Imidazole, Merck KGaA, Batch S6746923, Purity (GC): 99.8% (a/a)

APPLICATION DOSE AND EXPOSURE TIME
Fresh incubation medium was filled into the posterior compartment, while the surface of the cornea in the anterior compartment was treated with 750 µL of either the test item preparation, negative or positive control. The negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber, whereas the test item was applied to the anterior chamber with the open-chamber method. For this, the window locking-ring and the glass window were removed from the anterior chamber prior to treatment. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1°C for 240 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: none

REMOVAL OF TEST SUBSTANCE
After the incubation period, the negative and positive control preparations were removed from the anterior chamber without opening the chamber. The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Fresh incubation medium was replaced in both compartments prior to reading the opacity value after treatment. To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments.

- POST-EXPOSURE INCUBATION:
none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: An increased permeability of the cornea is indicative for an impairment of the integrity of the corneals’ epithelial cell layers. To determine the permeability of the treated corneas, fresh incubation medium was added to the posterior compartments and 1 mL of a fluorescein solution was administered to the anterior compartments. The corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes. The amount of fluorescein that crossed the cornea was measured spectrophotometrically in the medium from the posterior chamber.

- Others: Each cornea was observed visually and pertinent observations were recorded (e.g.tissue peeling, residual test chemical, non-uniform opacity patterns).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA: Decision criteria as indicated in the TG was used.
IVIS ≤ 3 results in no classification, IVIS >55 results in GHS-classification as “inducing serious eye damage, category 1”, IVIS between >3 and ≤55 results in ‘no prediction can be made’

Definition of Study Acceptance Criteria
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean (IVIS positive control: 81.7 – 132.9).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.4 – 3.2).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1
Value:
0.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.7 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.2).
- Acceptance criteria met for positive control: yes
After treatment with the positive control (20% Imidazole) the calculated IVIS was 101.0 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 81.7 – 132.9).
Therefore, the study fulfilled the acceptance criteria.

Any other information on results incl. tables


Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
0.9 -0.002
0.870
0.7
0.2
0.7
-0.004
0.640
0.5
-0.003
0.455
Positive control
20% Imidazole solution
59.0
2.671
99.065
101.0
9.3
63.9
3.150
111.150
64.4
1.897
92.855
Test item
Test item
-0.2
0.003
-0.155
0.6
1.0
1.8
-0.001
1.785
0.2
-0.001
0.185


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage.
Executive summary:

A study according OECD TG 437 was conducted to determine the eye hazard potential of the test item. The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.7 (study acceptance criteria range: -1.4 – 3.2). Treatment with the positive control (20% Imidazole) revealed an IVIS of 101.0 (study acceptance criteria range: 81.7 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment was 0.6 and, thus lower than 3, i.e.according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage.