[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:

17 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 in 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, vegetable-oil, polymd., esters with 1,2-hexadecanediol

Cas Number:

68910-99-6

Molecular formula:

Not applicable (i.e., UVCB substance)

IUPAC Name:

Fatty acids, vegetable-oil, polymd., esters with 1,2-hexadecanediol

Test material form:

other: paste

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Characteristics of donor animals: young cattle
- Storage, temperature and transport conditions of ocular tissue: eyes were collected and transported in physiological saline in a suitable container under cooled conditions
- indication of any existing defects or lesions in ocular tissue samples: 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was tested neat. An excessive amount was applied with a filter paper.

Duration of treatment / exposure:

10 ± 1 min

Duration of post- treatment incubation (in vitro):

120 ± 10 min

Number of animals or in vitro replicates:

3 per treatment group

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, containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (BASF-OP3.0, 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 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

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. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline

SOLVENT CONTROL USED
not applicable

POSITIVE CONTROL USED
ethanol (≥ 99.9%)

APPLICATION DOSE AND EXPOSURE TIME
an excessive amount of test item was applied to the corneas
750 µL of either the negative or positive control were applied to the corneas
exposure time: 10 ± 1 min

POST-INCUBATION PERIOD: yes, 120 ± 10 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test item were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM.

- POST-EXPOSURE INCUBATION:
After washing, the medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490) , TECAN Infinite® M200 Pro Plate Reader

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the OECD TG 437 were used.

ACCEPTANCE CRITERIA: 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

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

10 min

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

/ the experimentally determined mean IVIS score is -0.2

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes: The individual in vitro irritancy scores for the negative controls ranged from 0.5 to 1.7 (mean 1.1) and were therefore less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: yes: The individual positive control in vitro irritancy scores ranged from 46 to 50. The mean in vitro irritancy score of the positive control (ethanol) was 48 and within two standard deviations of the current historical positive control mean.

Any other information on results incl. tables

Table 2. Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity ^1	Mean Permeability ^1	Mean IVIS Score^1, 2
Negative control	1.2	-0.001	1.1
Positive control (ethanol)	22	1.778	48
Test item	-0.2	0.003	-0.2

¹  Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²  In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Applicant's summary and conclusion

Interpretation of results:

other: not irritant to the eye according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified
Under the conditions of the Bovine Corneal Opacity and Permeability (BCOP) Test the test substance does not possess eye irritating properties.