Dibenzyl sulphoxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.12. – 10.12.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

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:

Adopted 26th July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dibenzyl sulphoxide

EC Number:

210-668-7

EC Name:

Dibenzyl sulphoxide

Cas Number:

621-08-9

Molecular formula:

C14H14OS

IUPAC Name:

(phenylmethanesulfinylmethyl)benzene

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation:
- Particle size distribution:
- Mass median aerodynamic diameter (MMAD):
- Geometric standard deviation (GSD):
- Shape of particles:
- Surface area of particles:
- Crystal structure:
- Coating:
- Surface properties:
- Density:
- Moisture content:
- Residual solvent:
- Activation:
- Stabilisation:
- Other:

White solid powder
Storage: The test substance was stored in dry room in dark in closed container at the room temperature.
Content of water: 0.025 % w/w (volumetric Karl Fischer titration)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic

- Characteristics of donor animals (e.g. age, sex, weight):
The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

- Time interval prior to initiating testing: The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). All eyes used in the assay were from the same group of eyes collected on a specific day.

- indication of any existing defects or lesions in ocular tissue samples:
Only corneas from eyes free of defects (including increased opacity, scratched, and neovascularisation) were used.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Treatment protocol for non-surfactant solids was used. Non-surfactant solids are typically tested as solutions or suspensions at 20% concentration.
The test substance was tested as suspension prepared from test substance at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.

Open-chamber method was used, because the test substance was not applied by the help of micropipette.
The test substance (the test substance in quantity enough to completely cover the cornea) was applied by the help of spatula directly to the epithelial surface of the cornea. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.

VEHICLE
0.9% NaCl
- Lot/batch no. (if required): 145068131, B. Braun Melsungen AG, Germany

Duration of treatment / exposure:

4 hrs

Duration of post- treatment incubation (in vitro):

1.5 hr

Number of animals or in vitro replicates:

The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.
Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 3, 4, 13)
Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 2, 6, 10)
Negative control group (0.9% NaCl) –3 corneas (No. 7, 8, 9)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Selection criteria for eyes used in BCOP:
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.


Preparation: Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium.
Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively.
Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

QUALITY CHECK OF THE ISOLATED CORNEAS
From 19 eyes the 6 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 2, 3, 4, 6, 7, 8, 9, 10 and 13) and 4 eyes were superfluous.

NUMBER OF REPLICATES
Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 3, 4, 13)
Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 2, 6, 10)
Negative control group (0.9% NaCl) – 3 corneas (No. 7, 8, 9)

NEGATIVE CONTROL USED
0.9% NaCl
SOLVENT CONTROL USED (if applicable)
0.9% NaCl
POSITIVE CONTROL USED
20% Imidazole

APPLICATION DOSE AND EXPOSURE TIME
Treatment protocol for non-surfactant solids was used. Non-surfactant solids are typically tested as solutions or suspensions at 20% concentration.
The test substance was tested as suspension prepared from test substance at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.
The test substance (the test substance in quantity enough to completely cover the cornea) was applied by the help of spatula directly to the epithelial surface of the cornea.
Exsposure time. 4 h

TREATMENT METHOD: Open-chamber method was used, because the test substance was not applied by the help of micropipette. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.

POST-INCUBATION PERIOD: 1.5 hr

REMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATION
After the exposure period, the test substance the negative control, or the positive control substance were removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale
- Corneal permeability:
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
≥ 55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

2.72

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The value of IVIS for positive control (20% Imidazole) obtained during the study was 76.20.
This value is within the acceptance limit (one standard deviation of the current historical mean), so the study is considered acceptable.

The value of opacity for negative control (0.9% NaCl) obtained during the study was 1.67 and value of permeability was 0.011.
The values obtained during this study not exceeded upper limits, so the study is considered acceptable.

Any other information on results incl. tables

The In Vitro Irritancy Score (IVIS) was computed according the following formula:

IVIS = mean opacity value + (15 x mean permeability OD490 value)

Group	IVIS calculation	Result
NC (0.9% NaCl)	1.67 + 15 x 0.011	1.84
PC (20% Imidazole in 0.9% NaCl)	48.66 + 15 x 1.836	76.20
EXP (Dibenzylsulfoxide)	2.66 + 15 x 0.004	2.72

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro Irritancy Score (IVIS) for Dibenzylsulfoxide was 2.72.
This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is:
No category.

Executive summary:

The test substance, Dibenzylsulfoxide, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 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, Adopted 26th July 2013.

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Open-chamber method was used, because the test substance was not applied by the help of micropipette.

Treatment protocol for non-surfactant solids was used. The test substance was tested as suspension prepared from test substance at 20% concentration in a 0.9% sodium chloride solution.

The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Dibenzylsulfoxide was 2.72.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.