Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Oct 2016 - 06 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(5-Ethyl-tetrahydropyran-2-yl)-phenol
EC Number:
921-114-5
Cas Number:
1173761-32-4
Molecular formula:
C13 H18 O2
IUPAC Name:
4-(5-Ethyl-tetrahydropyran-2-yl)-phenol
Test material form:
solid

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age at study initiation: 20-43 months
- Corneal diameter: 25 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Volume applied: 150 mg/750 µL
- Concentration: 20% (w/v) in a 0.9% NaCl solution

VEHICLE
- Volume applied: 750 µL

Duration of treatment / exposure:
240 minutes at 32 +/- 1 °C
Number of animals or in vitro replicates:
Three corneas were used per group (negative control, positive control and test item group).
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20% solution were applied for 240 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:at least 3 times with washing medium

- POST-EXPOSURE INCUBATION: in fresh medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: baseline opacity was determined with a calibrated opacitometer, the light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes, amount of fluorescein that crossed the cornea was measured spectrophotometrically 490 nm

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
- The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean(IVIS positive control: 75.3 - 120.2).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.2 - 5.3).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.


Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
replicate 1
Run / experiment:
1
Value:
95.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
replicate 2
Run / experiment:
1
Value:
99.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
replicate 3
Run / experiment:
1
Value:
97.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
1
Value:
97.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: yes


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Any other information on results incl. tables

Table 1 Opacity, permeability and IVIS results

 

Opacity

Permeability

IVIS

per cornea

per group
(mean value)

Standard
deviation

Negative
control

0.9% sodium
chloride solution

3.0

0.000

3.000

1.5

1.8

1.9

0.001

1.915

-0.5

-0.001

-0.515

Positive
control

20 % Imidazole in 0.9 % NaCl solution

69.6

2.422

105.930

105.4

8.6

49.1

3.168

96.620

72.9

2.720

113.700

Testitem

Test item 20 % in 0.9 % NaCl solution

53.1

2.853

95.895

97.7

1.7

44.4

3.661

99.315

54.3

2.906

97.890

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the conditions of the present study the test item induces serious eye damage.
Executive summary:

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro lrritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: - 1.4 - 3.4). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 105.4 (study acceptance criteria range: 78.6 - 135.0). Therefore, the study fulfilled the acceptance criteria.

The IV IS obtained after treatment with the test item was 97.7 and, thus, higher than 55, i.e. according to OECD 437 the test item induces serious eye damage (UN GHS: Category 1).