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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Feb 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
Jun 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,6-anhydro-β-D-glucose
EC Number:
207-855-0
EC Name:
1,6-anhydro-β-D-glucose
Cas Number:
498-07-7
Molecular formula:
C6H10O5
IUPAC Name:
(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol
Constituent 2
Chemical structure
Reference substance name:
Glycollaldehyde
EC Number:
205-484-9
EC Name:
Glycollaldehyde
Cas Number:
141-46-8
Molecular formula:
C2H4O2
IUPAC Name:
2-hydroxyacetaldehyde
Constituent 3
Reference substance name:
Organic acids
IUPAC Name:
Organic acids
Constituent 4
Reference substance name:
Ketones
IUPAC Name:
Ketones
Constituent 5
Reference substance name:
Phenols
IUPAC Name:
Phenols
Constituent 6
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Constituent 7
Reference substance name:
Oligomers of sugars and anhydrosugars
IUPAC Name:
Oligomers of sugars and anhydrosugars
Test material form:
liquid: viscous

Test animals / tissue source

Species:
cattle
Strain:
other: Bos rimigenius Taurus (fresh bovine corneas) p
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Industri- estraße 42, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test material:
-amount applied: 750 μL
- concentration: neat
Duration of treatment / exposure:
Exposure time: 10 minutes at 32 ± 1 °C.
After thorough rinsing the anterior chambers with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chambers were filled with cMEM without phenol red and the cornea holders were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time the final opacity value of each cornea was recorded.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
Preparation of Test System
After having carefully cleaned and sterilized the cornea holders, they were kept in the incu bation chamber at 32 ± 1 °C.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used.
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. The formation of bubbles was prevented.
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the cornea holder in an opacitometer and recording the illuminance (unit: LUX).
None of the corneas showed an opacity greater than seven opacity units; therefore, all corneas were used.

Preparations
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bi- carbonate.

Application
For each treatment group (negative control solution, test item or positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL of negative control solution, 750 μL of test item or 750 μL of positive control were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the controls, the following treatment procedure was performed:
Closed Chamber Method:
The respective substance (negative control solution, test item solution or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the anterior holder on the cornea. The controls and the test item were given on the epithelium that the cornea was evenly covered.
According to the characteristics of the test item, the following treatment procedure was per- formed:

Open Chamber Method:
In order to apply the test item, the window-locking ring and glass window of the anterior chamber of each cornea holder was unscrewed to remove the glass disc.
750 μL of the test item was applied directly onto the cornea. After dosing, the glass windows were replaced on the anterior chambers to recreate a closed system.

Permeability test
After the recording of the final opacity values, the cMEM without phenol red was removed from both chambers of each cornea holder. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM. Then 1 mL sodium fluorescein solution was added to the front chamber of each cornea holder for the detection of permea- bility of the corneas.
For the controls and the test item a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a hori- zontal position. After incubation, the content of each posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Three replicates
Value:
57.59
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean of three replicates
Value:
40.61
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability
Run / experiment:
three replicates
Value:
1.131
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The mean IVIS of the negative control has to show an IVIS ≤ 3.

The validity criteria
Mean IVIS of negative control HBSS, criterion: ≤3, result: 1.24 -->ok
Mean IVIS of positive control DMF undiluted, criterion: between 57.92 and 139.81, result: 107.83 -->ok

All validity criteria were met. The values for the negative and positive control were within the range of historical data of the test facility (see Annex 2, page 22). Therefore, the test system was acceptable.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item Pyrolytic Sugar is found to induce serious eye damage in the Bovine Corneal Opacity and Permeability (BCOP) Test.
Executive summary:

The test was performed in accordance to the OECD TG 437 and in compliance to GLP.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item Pyrolytic Sugar was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was 1.24.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 107.83.

Under the conditions of this study, the test item Pyrolytic Sugar induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 57.59.

According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 55 in- duces serious eye damage, that should be classified as UN GHS Category I.

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