1-fluoro-3-(trans-4-propylcyclohexyl)benzene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 FEB 2015 - 19 MAR 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:

1-fluoro-3-(trans-4-propylcyclohexyl)benzene

EC Number:

443-100-3

EC Name:

1-fluoro-3-(trans-4-propylcyclohexyl)benzene

Cas Number:

138679-81-9

Molecular formula:

Hill formula: C15H21F CAS formula: C15H21F

IUPAC Name:

1-fluoro-3-[(1s,4r)-4-propylcyclohexyl]benzene

Test material form:

liquid

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: 11 - 28 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) and in the incubation medium (5 mL/500 mL)

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):

120 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
0.9 % sodium chloride solution

POSITIVE CONTROL USED
N,N-dimethylformamide (undiluted)

APPLICATION DOSE AND EXPOSURE TIME
750 µL for 10 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: 120 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The corneal surface was washed three times with wash medium. Afterwards, incubation medium was used as final rinse to ensure removal of the phenol red from both chambers.

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 of three corneas

Value:

-0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

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: An in vitro study was performed to determine the eye hazard potential of the two test items Tween 20 and Trichloroacetic acid by means of the BCOP (Bovine Corneal Opacity and Permeability Assay) to check the accuracy and reliability of the test method as recommended by OECD Guideline 437. The IVIS obtained after treatment with Tween 20 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 Trichloroacetic acid was 268.5 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 2.6 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 - 5.7).
- Acceptance criteria met for positive control: After treatment with the positive control (N,N-dimethylformamide) the calculated IVIS was 107.7 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 79.9 - 120.2).

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Executive summary:

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay) according to OECD Guideline 437 and following GLP. Therefore, the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide 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 test item, positive or negative control were applied on the corneas and incubated for 10 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 2.6. Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 107.7. Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was -0.4 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.