Benzaldehyde, 4-[(3-fluorophenyl)methoxy]-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 OCT 2014 - 08 JAN 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Unwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden (18 November 2014)

Test material

Test material information

Constituent 1

Reference substance name:

4-[(3-fluorophenyl)methoxy]benzaldehyde

EC Number:

613-982-6

Cas Number:

66742-57-2

Molecular formula:

C14H11FO2

IUPAC Name:

4-[(3-fluorophenyl)methoxy]benzaldehyde

Test material form:

solid: crystalline

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals (e.g. age, sex, weight):
age: 18 - 26 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
transport medium: HBSS (Hanks' Balanced Salt Solution)
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. In addition, an opacity measurement before treatment was performed. Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity of >7 opacity units were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS)

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 150 mg/750 µL (20 % (w/v))

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9 % sodium chloride

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (prewarmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, 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 form.

QUALITY CHECK OF THE ISOLATED CORNEAS
Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
solvent control serves as negative control

SOLVENT CONTROL USED (if applicable)
yes, 0.9% sodium chloride solution

POSITIVE CONTROL USED
yes, 20% (w/v) Imidazole solution (in 0.9% sodium chloride solution)

APPLICATION DOSE AND EXPOSURE TIME
50 mg/750 µL (20 % (w/v)) for 240 min

TREATMENT METHOD: closed chamber for positive and negative control, open chamber for test item treatment

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. Using a syringe, the corneas treated with the test item were gently rinsed with wash medium. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity was determined with an opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value was recorded in a table and converted into an opacity value. The opacitometer was calibrated as described in the manual and the opacity of each cornea was determined by reading each holder placed in the photoreceptor compartment of the opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): visual inspection of the corneas after treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the OECD Guideline 437 were applied. The IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the following:
≤ 3 No Category (UN GHS)
< 3; ≤ 55 No prediction can be made
> 55 Category 1 (UN GHS)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was demonstrated in a study with 2 of the 13 proficiency chemicals recommended in OECD Guideline 437. The IVIS obtained after treatment with Phenylbutazone was 0.9 and, thus, lower than 3. Therefore, the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category). The IVIS obtained after treatment with (-)-Dibenzoyl-L-tartaric acid was 158.8 and, thus, higher than 55. Therefore, the test item requires classification for serious eye damage (UN GHS classification: Category 1).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.3 - 3.3).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 98.0 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 74.7 - 138.3).

Any other information on results incl. tables

A table of the results is attached under 'Attached background material'.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The IVIS obtained after treatment with the test item was 2.4 and, thus, lower than 3. Therefore, test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Executive summary:

A BCOP (Bovine Corneal Opacity and Permeability Assay) according to OECD Guideline 437 following GLP was performed with the test item. 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 Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1. Treatment with the positive control (20% Imidazole) revealed an IVIS of 98.0. Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was 2.4 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.