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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 January 2019 - 10 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed under GLP and all relevant validity criteria were met

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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:
2017
Deviations:
no
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:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)
EC Number:
680-227-5
Cas Number:
71449-78-0
Molecular formula:
C24H19F6S2Sb
IUPAC Name:
Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)
Test material form:
solid: particulate/powder
Remarks:
white
Details on test material:
- Storage condition of test material: Room temperature, in the dark

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Corneas from bovine eyes were obtained from a local abattoir.
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, completely immersed in Hanks Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin at 100 IU/mL and streptomycin at 100 µg/mL added to Hanks Balanced Salt Solution during transportation.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Duration of treatment / exposure:
240 minutes.
Observation period (in vivo):
n/a
Duration of post- treatment incubation (in vitro):
90 minutes.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


QUALITY CHECK OF THE ISOLATED CORNEAS

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

NUMBER OF REPLICATES

3 per treatment group

NEGATIVE CONTROL USED

The negative control substance was 0.9% sodium chloride solution (Batch; 18/10BB1B, exp: 10 April 2019) supplied by Baxter.

SOLVENT CONTROL USED (if applicable)

N/A

POSITIVE CONTROL USED

The positive control substance was Imidazole >99% (Batch; 162411, exp: 01 August 2021) Supplied by Fisher Scientific.

APPLICATION DOSE AND EXPOSURE TIME

0.75 mL applied to each cornea and incubated at 32 ± 1 °C for 240 minutes.

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION: 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Labtech LT-4500 microplate reader
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD492)
- Others (e.g, pertinent visual observations, histopathology): (please specify) The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
3 runs
Value:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: n/a

Any other information on results incl. tables

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements & IVIS Score

Treatment

 

Cornea No.

Opacity

Permeability

In Vitro Irritancy Score

 

 

Pre-Treatment

Post-Treatment

Post-Treatment - Pre-Treatment

Corrected Value

 

Corrected Value

 

Negative Control

1

5

5

0

 

0.003

 

 

2

4

5

1

 

0.005

 

 

3

5

6

1

 

0.004

 

 

 

 

 

0.7*

 

0.004¨

 

0.7

Positive Control

 

6

5

75

70

69.3

1.264

1.260

 

7

5

79

74

73.3

1.490

1.486

 

8

3

68

65

64.3

1.810

1.806

 

 

 

 

 

69.0!

 

1.517!

91.8

Test Item

10

4

4

0

0.0

0.054

0.050

 

11

3

6

3

2.3

0.015

0.011

 

12

6

7

1

0.3

0.003

0.000

 

 

 

 

 

0.9·

 

0.020·

1.2

OD = Optical density

* = Mean of the post-treatment & pre-treatment values

" = Mean permeability

! = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that under the condition of this study, the test substance produced an IVIS score of 1.2 and therefore no categorisation for irritation could be made and the substance does not meet the criteria for classification in accordance with UN GHS or EU CLP.
Executive summary:

OECD 437 (2019) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using CPI-110A in accordance with 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" (2017).

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for


240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability), were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 492 nanometers (OD492). The mean corrected opacity reading and permeability readings for the test item were 0.9 and 0.020, resulting in an In Vitro Irritation Score (IVIS) of 1.2

It was concluded that under the condition of this study, the test substance produced an IVIS score of 1.2 and therefore no categorisation for irritation could be made and the substance does not meet the criteria for classification in accordance with  UN GHS or EU CLP.