[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:

Experimental phase: 24 November 2017. Report issues: 17 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Molecular formula:

not applicable as UVCB

IUPAC Name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 ml of either test substance, negative control or positive control covering the anterior compartment of the eye.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Two hours

Number of animals or in vitro replicates:

Three replicates (cornea) per treatment (test substance, negative control, positive control)

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS : Yes

NUMBER OF REPLICATES : three per treatment

NEGATIVE CONTROL USED: Yes, Saline (0.9% NaCl in deionised water)

SOLVENT CONTROL USED: No

POSITIVE CONTROL USED : Yes, 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME : 10 minutes

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After expsoure, the test and control substances were rinsed off from the application side with saline, fresh cMEM was then added into the anterior compartment.

POST-INCUBATION PERIOD: Yes, two-hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom France))
- Corneal permeability: passage of sodium fluorescein dye measured with microtitre plate reader at 490nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test substance Exposure Cornea 1

Value:

21.19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance Exposure Cornea 2

Value:

11.98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance Exposure Cornea 3

Value:

14.49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS Score for Test Substance

Value:

15.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Relative to the negative control, the test substance caused an increased in corneal opacity. The calculated mean IVIS was 15.89 which is below the threshold for serious eye damage (IVIS > 55).

Any other information on results incl. tables

Test Results (10 Minute Treatment time)

Test Group	Opacity Value = Difference (t130 -t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	In vitro Irritancy Score
		Mean		Mean
Negative Control	1	0.67	0.061	0.078	1.92	1.86	No category
	1		0.068		2.02
	0		0.109		1.64
Positve Control	56.33*		1.703*		81.87	87.23	Category 1
	61.33*		1.652*		86.11
	73.33*		1.359*		93.71
Test Substance	21.33*		-0.009*		21.19	15.89	No prediction can be made
	12.33*		-0.023		11.98
	14.33*		0.011		14.49

*corrected values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the experimental conditions reported, the test substance does not cause serious eye damaging (i.e. it is not classified GHS Category 1 for eye effects).

Executive summary:

Introduction

This in vitro study was performed to assess the corneal damage potential of the test substance by means of the BCOP assay using fresh bovine corneae. The test was designed to meet the requirements of OECD Guideline for Testing of Chemicals 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 (July, 2013).

Method

After a first opacity measurement of the fresh bovine corneae (t₀), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards,opacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

Results

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability ofthe corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS(Cat 1)).

 

Relative to the negative control, the test substance caused an increase of the corneal opacity. The calculated mean in vitro irritancy score was 15.89 which is below the threshold for classification for serious eye damage (IVIS > 55) according to CLP/EPA/GHS.

 

Conclusion

In conclusion, under the experimental conditions reported, the test substance does not cause serious eye damaging (i.e. it is not classified CLP/EPA/GHS Category 1 for eye effects).