[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study. Deviation: When a 20% w/v formulation in saline was prepared the test item was considered to be unsuitable for dosing. Therefore in accordance with the methods described within the OECD BCOP test guideline, the test item was applied neat onto the corneal surface.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

When a 20% w/v formulation in saline was prepared the test item was considered to be unsuitable for dosing. Therefore in accordance with the methods described within the OECD BCOP test guideline, the test item was applied neat onto the corneal surface.

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C18, branched and linear

Molecular formula:

Not applicable as UVCB

IUPAC Name:

Amides, C18, branched and linear

Test material form:

solid: pellets

Details on test material:

- Name of test material (as cited in study report): Amides, C18, branched and linear
- Physical state: extremely pale yellow granular solid
- Lot/batch No.: 202637
- Expiration date of the lot/batch: 20 April 2016
- Stability under test conditions: stable
- Storage condition of test material: at RT, protect from light
- Analytical purity: 100%

Test animals / tissue source

Species:

other: cattle

Strain:

other: bovine eyes from young cattle

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an invitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Transport medium and temperature conditions: The eyes were placed in HBSS supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL) and transported to the test facility over ice packs on the same day of slaughter.

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Isolated corneas were mounted in cornea holders
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea
- Test medium and temperature conditions used in the cornea holder: cMEM (Eagle’s Minimum Essential Medium); prior to use: prewarmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C

DETERMINATION OF THE INITIAL OPACITY
Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: number of eyes for the negative control: 3; number of eyes for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 0.3 g

NEGATIVE SUBSTANCE
- Substance: sodium chloride solution
- Concentration (if solution): 0.9% (w/v) NaCl solution in deionised water

POSITIVE SUBSTANCE
- Substance: Imidazole
- Concentration (if solution): 20% (w/v) Imidazole in 0.9% (w/v) NaCl solution in deionised water

Duration of treatment / exposure:

4 h

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

number of eyes for the test item: 3

Details on study design:

TEST CONDITIONS
- Open-Chamber method: After dosing, the glass window was replaced on the anterior chamber to recreate a closed system. Corneas were exposed for 4 h with the test substance or the controls. The glass window from the anterior chamber was removed prior to treatment. The controls or test substance were applied directly to the epithelial surface of the cornea.

POSTEXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the cornea was rinsed three times.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: complete MEM

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: Directly after refilling fresh cMEM without phenol red in the anterior chamber the final opacity was measured.

DETERMINATION OF THE CORNEAL PERMEABILITY
- Method: The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution. The dosing holes were plugged and the holders incubated, anterior chamber uppermost. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 492 nm recorded as optical density (OD492).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL; dissolved in cMEM)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: OD492 of a 360 μL aliquot was determined in a 96-well plate
- Specification of the spectrophotometer: Anthos 2001 microplate reader

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Three experiments run with an exposure time of 4 hours

Value:

ca. 0

Negative controls validity:

valid

Remarks:

Mean opacity change of NC 1.0

Positive controls validity:

valid

Remarks:

Mean opacity value if 56.0

Remarks on result:

no indication of irritation

Remarks:

Please see the "Any other information on results incl. tables" for more detailed info on results.

Results 2

Irritation parameter:

other: Permeability values (optical density (OD) at 492 nm)

Run / experiment:

Three experiments run with an exposure time of 4 hours

Value:

ca. 0.019

Negative controls validity:

valid

Remarks:

Mean OD492 value is 0.073

Positive controls validity:

valid

Remarks:

Corrected mean OD492 value is 1.461

Remarks on result:

no indication of irritation

Remarks:

Please see the "Any other information on results incl. tables" for more detailed info on results.

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

IVIS

Run / experiment:

Three experiments run with an exposure time of 4 hours

Value:

ca. 0.3

Negative controls validity:

valid

Remarks:

Mean IVIS 2.1

Positive controls validity:

valid

Remarks:

Mean IVIS 77.9

Remarks on result:

no indication of irritation

Remarks:

Please see the "Any other information on results incl. tables" for more detailed info on results.

Any other information on results incl. tables

Table 1: Opacity values

Parameter	Initial opacity	Final opacity	Opacity change	Mean opacity change of NC	Corrected opacity change		Mean opacity value
Negative control	1	2	1	1.0		-	-
	1	2	1
	1	2	1
Test substance	1	2	1	-	0.0		0.0
	1	2	1		0.0
	2	2	0		0.0
Positive control	0	60	60	-		59	56.0
	0	59	59			58
	3	55	52			51

Table 2: Permeability values (optical density (OD) at 492 nm)

Parameter	OD492	Mean OD492  value	Corrected OD492	Corrected Mean OD492  value
Negative control	0.076	0.073	-	-
	0.073
	0.070
Test substance	0.095	-	0.022	0.019
	0.100		0,027
	0.081		0.008
Positive control	1.533	-	1.460	1.461
	1.536		1.463
	1.533		1.460

Table 3: In-Vitro Irritancy Score (IVIS) values

	Mean IVIS
Negative control	2.1
Test substance	0.3
Positive control	77.9

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified