[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:

weight of evidence

Study period:

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)

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Molecular formula:

C32H58O10

IUPAC Name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Constituent 2

Reference substance name:

not yet assigned

Molecular formula:

C50H88O16 C51H90O16 C52H92O16

IUPAC Name:

not yet assigned

Constituent 3

Reference substance name:

2,3-epoxypropyl neodecanoate

EC Number:

247-979-2

EC Name:

2,3-epoxypropyl neodecanoate

Cas Number:

26761-45-5

Molecular formula:

C13H24O3

IUPAC Name:

(oxiran-2-yl)methyl 2,2-dimethyloctanoate

Test material form:

liquid: viscous

Details on test material:

Former EC 615-318-0 / CAS: 716336-43-5: changed to EC 825-846-5 / CAS 876528-25-5

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 °C (± 1 °C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

approximately 2 hours

Number of animals or in vitro replicates:

3 corneas per tested material

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 °C (± 1 °C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
yes

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: NaOH (1 %)

APPLICATION DOSE AND EXPOSURE TIME
the liquid test materials were applied pure for 10 min

TREATMENT METHOD:
closed chamber method

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer (BASF OP3.0). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1 mL of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Test item formulations that cause an IVIS value > 55 are classified as seriously damaging the eye (UN GHS Cat 1). Test item formulations that cause IVIS values of ≤ 55 are considered as not seriously damaging the eye (not UN GHS Cat 1).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

mean

Value:

-0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

tested materials  opacity values per cornea  permeability values per cornea  IVIS per cornea  IVIS mean  negative control (3 corneas) 4.5, 4.2, 4,1  0.010, 0.007, 0.005  4.6, 4.3, 4.2  4.4 (SD 0.2)  positive control (3 corneas) 175.9, 143.7, 188.2  1.679, 1.249, 1.785 201.1, 162.5, 215.0  192.9 (SD 27.2)  test item  1.8, -0.3, -3.7  0.000, 0.003, 0.003  1.8, -0.2, -3.6  -0.7 (SD 2.7)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

The test item was tested in the BCOP test according to OECD TG 437. For determination of corneal damage corneal opacity as well as tissue permeability was measured after a 10 min incubation time. Based on these data, in comparison to the negative control, the In Vitro Irritancy Score (IVIS) value was calculated with -0.7 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage.