Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 17 April 2018. Experimental completion date: 17 April 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
October 09, 2017
Deviations:
no
GLP compliance:
yes
Remarks:
Analyses conducted to support the information cited in the Certificate of Analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a Sponsor quality system.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BZA11014
- Expiration date of the lot/batch: 21 April 2018
- Manufacturing date: 21 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature under nitrogen
- Not stable at higher temperatures

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. The test item was tested neat.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
10 +/- 1 minutes
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1ºC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1ºC.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
Ethanol.

APPLICATION DOSE AND EXPOSURE TIME
The test item was tested neat (colourless liquid) for 10 +/- 1 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 120 +/- 10 minutes

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = ((I0/I) - 0.9894) / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Each cornea was inspected visually for dissimilar opacity patterns.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490) . Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1ºC. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

DECISION CRITERIA: The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
<=3 No Category
>3 <= 55 No prediction can be made
>55 Category 1
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
>= -3 - <= 1
Negative controls validity:
valid
Remarks:
Opacity and permeability values less than the upper limits of the laboratory historical range
Positive controls validity:
valid
Remarks:
Value was 53 and within two sandard deviations of the current historical positive control mean
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Remarks:
Permeability values
Value:
>= 0.001 - <= 0.004
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The corneas treated with Thio Isocyanate Adduct showed opacity values ranging from -3.0 to 1.0 and permeability values ranging from 0.001 to 0.004. The corneas were translucent after the 10 minutes of treatment with Thio Isocyanate Adduct. No pH effect of the test item was
observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.9 to 1.1 after 10 minutes of treatment with Thio Isocyanate Adduct.

OTHER EFFECTS:
- The corneas treated with the negative control item were translucent after the 10 minutes of treatment.
- The corneas treated with the positive control item were turbid after the 10 minutes of treatment.
- The corneas treated with the test item were translucent after the 10 minutes of treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores:

 Treatment Mean opacity Mean permeability  Mean in vitro irritation score 
 Negative control  1.8 0.000 1.8 
 Positive control 18 2.319 53 
 Test item -1.1  0.003  -1.1 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since the Thio Isocyanate Adduct induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

The study was performed to evaluate the eye hazard potential of Thio Isocyanate Adduct as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The eye damage of Thio Isocyanate Adduct was tested through topical application for 10 minutes. The study procedures were based on OECD guideline 437 and according to GLP. The test item was applied as it is (750 μL) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 53 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Thio Isocyanate Adduct did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 after 10 minutes of treatment. In conclusion, since Thio Isocyanate Adduct induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.