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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-18 to 2019-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Deviations:
yes
Remarks:
The positive control is 2-Ethoxyethanol (purity: 99%) since the laboratory historical control data are established with this chemical.
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate
EC Number:
949-569-5
Molecular formula:
C12H22O2
IUPAC Name:
Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate
Test material form:
liquid

Test animals / tissue source

Species:
other: Freshly isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals:
- Characteristics of donor animals: 14 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: 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 and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test item, positiv or negativ control
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
3 replicates per test group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of 14 month old donor cattle were collected from the abattoir. All eyes were carefully examined macroscopically for defects. 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. Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0). At the end of the incubation period, the basal opacity was determined (t0). Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

NUMBER OF REPLICATES : three

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

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

APPLICATION DOSE AND EXPOSURE TIME
The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes (± 30 seconds).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 4 times - 3 times with EMEM containing phenol red and 1 time with cMEM without phenol red
- POST-EXPOSURE INCUBATION: The corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). In the second step of the assay, permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France); The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value.
- Corneal permeability: the optical density at 490 nm (OD490) was measured with a microplate reader (Versamax® Molecular Devices)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Depending on the score obtained, the test item was classified according to OECD guideline 437.
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
test item group
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Results after 10 Minutes Treatment Time

Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

1

0.33

0.094

0.078

2.41

1.51

No category

0

0.072

1.08

0

0.069

1.04

Positive Control

87.67*

1.417*

108.92

99.81

Category 1

75.67*

1.059*

91.55

82.67*

1.087*

98.97

Test Item

-0.33*

-0.032*

0.00#

0.00

No category

-0.33*

-0.030*

0.00#

-0.33*

-0.024*

0.00#

 

With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.51).

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 99.81) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item was tested undiluted. Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.00 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study the test item is not an eye irritant.
Executive summary:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was conducted. After a first opacity measurement of the fresh bovine cornea (t0), the undiluted test item, the positive control, and the negative control were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 °C. After the incubation phase the test item, the positive control, and the negative controls were each rinsed from the cornea. Further, the corneae were incubated for another 120 min at 32 °C in a vertical position. Afterwards, opacity was measured a second time (t130). Permeability of the cornea was also determined after opacity measurements.

The acceptance criteria for the negative and positive control were met. With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.51). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 99.81) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

In comparison to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.00 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified as serious eye damaging or as eye irritating.