2-cyclohexylethyl 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:

2018

Report date:

2018

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-cyclohexylethyl acetate

EC Number:

244-543-3

EC Name:

2-cyclohexylethyl acetate

Cas Number:

21722-83-8

Molecular formula:

C10H18O2

IUPAC Name:

2-cyclohexylethyl acetate

Specific details on test material used for the study:

6.1 Test Item
Designation in Test Facility: 17112908G
Date of Receipt: 29. Nov. 2017
Condition at Receipt: Room temperature, in proper conditions
6.1.1 Specification
The following information concerning identity and composition of the test item was provid-ed by the sponsor.
Name Cyclohexyl ethyl acetate
Batch no. CP014-171101
Appearance colorless and transparent liquid
Composition Cyclohexyl ethyl acetate
Purity 99.85%
Homogeneity homogeneous
Expiry date 30. May. 2019
Storage Room temperature (20 ± 5 °C)

The following additional information, provided by the sponsor too, was relevant to the
conduct of the study, according to OECD 437:
CAS No. 21722-83-8
EINECS-No. 244-543-3
Chemical Class not stated
Volatility unknown
pH-value unknown
Stability unknown
Solubility unknown

6.1.2 Storage
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C).
6.1.3 Preparation
The test item is a liquid substance. It was tested directly, without dilution or preparation of a solution.

Test animals / tissue source

Species:

other: Bos primigenius Taurus (fresh bovine corneas)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 30 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Details on study design:

After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, test item and positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:
 
7.3.1 Closed Chamber Method
The respective substance (negative control solution, test item or positive control) was ap-plied by pipetting 750 µL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded.

7.3.2 Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the closed chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 4.81

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Parameter C riterion Found Assessment
IVIS of negative control
HBSS ≤ 3 1.01 ok
IVIS of positive control
DMF undiluted 36.44 – 149.99 100.81 ok

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.