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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:
27 Feb 2017 - 27 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methyldecan-1-al
EC Number:
242-745-6
EC Name:
2-methyldecan-1-al
Cas Number:
19009-56-4
Molecular formula:
C11H22O
IUPAC Name:
2-methyldecanal
Test material form:
liquid
Details on test material:
Appearance: clear, colourless liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: corneae from at least 9 month old donors

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): 0.75 mL
- Concentration (if solution): N/A

VEHICLE -N/A
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
x3 replicates per test group
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: 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 transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin). The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, 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.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and 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. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
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 basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

QUALITY CHECK OF THE ISOLATED CORNEAS: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NUMBER OF REPLICATES: 3 per group

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

SOLVENT CONTROL USED (if applicable) N/A

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

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes incubation

TREATMENT METHOD: [closed chamber / open chamber] Not specified

POST-INCUBATION PERIOD: Yes - two hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 1 step
- POST-EXPOSURE INCUBATION: After exposure of the corneae to the test groups, corneae were rinsed and incubated for a further 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 the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) The permeability endpoint was measured, as an indication of the integrity of the epithelial cell sheets.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
>= 0.67 - <= 1.67
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
>= 0 - <=1
Positive controls validity:
valid
Remarks:
>=74.67 - <=95.67
Remarks on result:
no indication of irritation
Remarks:
IVIS score: <= 3
Irritation parameter:
in vitro irritation score
Value:
1.02
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.44
Positive controls validity:
valid
Remarks:
98.08

Any other information on results incl. tables

Table 1 demonstrates the results of the test groups after 10 minutes incubation.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.44). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.08) 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 an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.02 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Table 1. Results after 10 minutes incubation 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

0.069

0.074

2.04

1.44

Not categorised

0

0.081

1.22

0

0.072

1.08

Positive control

95.67*

0.643*

105.31

98.08

Category 1

84.67*

0.890*

98.02

74.67*

1.063*

90.91

Test item

0.67*

-0.009*

0.53

1.02

Not categorised

1.67*

0.011*

1.83

0.67*

0.002*

0.70

*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The calculated mean in vitro irritancy score was 1.02. Therefore, the test item is not categorized (GHS).



Executive summary:

The test item was assessed for eye irritation as per OECD Guideline 437, 26 July 2013.

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.

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.02 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified for eye irritation.

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