1,3-dimethyl-3-phenylbutyl acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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:

no

Test guideline 2

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dimethyl-3-phenylbutyl acetate

EC Number:

268-407-8

EC Name:

1,3-dimethyl-3-phenylbutyl acetate

Cas Number:

68083-58-9

Molecular formula:

C14H20O2

IUPAC Name:

4-methyl-4-phenylpentan-2-yl acetate

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The purpose of this study was to assess the possible corneal damage potential when fresh bovine corneae are exposed to the test item. This study should provide a rational basis for risk assessment in humans.
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: not reported, but sets of three corneae were used for treatment with the test item and the negative and positive controls.
- Characteristics of donor animals (e.g. age, sex, weight): at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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 until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: 1 % (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) was used during storing freshly isolated eyes.

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

Duration of treatment / exposure:

10 minutes

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Freshly isolated bovine eyes were carefully examined macroscopically for defects and eyes showing 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 as described in the guideline.

QUALITY CHECK OF THE ISOLATED CORNEAS: After mounting the corneas in the holder, 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.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Saline (0.9% NaCl in deionised water, using ultrasonic technique)

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

APPLICATION DOSE AND EXPOSURE TIME: The test item was applied in a volume of 0.75 mL on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for ten minutes.

TREATMENT METHOD: the test item or negative or positive control was applied into the anterior compartment on the surface of the corneae.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 1
- POST-EXPOSURE INCUBATION: After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The test item was classified into the following category according to OECD guideline 437:
≤ 3: No category
>3 - ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

1.81

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

3.93

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

1.89

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

2.54

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS =1.09).
- Acceptance criteria met for positive control: yes, clear opacity and distinctive permeability of the corneae observed (mean IVIS = 83.62) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The calculated mean in vitro irritancy score for the test item was 2.54. The test item is not classified as eye irritant according to GHS.

Executive summary:

An in vitro study was performed according to the OECD 437 guideline to assess the corneal damage potential of test item by means of the BCOP assay using fresh bovine corneae.

The undiluted test item, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, treatment and controls were each rinsed and the corneae were incubated for

another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured. 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 a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 2.54. The test item is not classified as eye irritant according to GHS.