1-Ethoxy-2,3-difluor-4-[4-(trans-4-propylcyclohexyl)-1-cyclohexen-1-yl]-benzene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-09-24 to 2007-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Hen's egg test on the chorioallantoic membrane (HET-CAM) for irritating potential

Version / remarks:

according to Lüpke (Fd. Chem. Tox. 1985; 23 (2):287-291), with modifications described by Kalweit (Toxic. in vitro 1990; 4 (4/5):702-706)

Principles of method if other than guideline:

During the second part of the 1980's, with a growing number of chemicals to be tested and due to an increasing awareness towards the need to protect animals from painful experiments, the hen's egg chorioallantoic membrane test (HET-CAM) was developed. The chorioallantoic membrane (CAM) of fertilized and incubated hen eggs provides a suitable model for membranes with an intact and functional vascular system. The CAM has no nerve fibers in the early stages of development and is regarded as being insensitive to pain. For these reasons, the HET-CAM is believed to be a reliable alternative to the primary eye irritation test in rabbits, which does not conflict with ethical and legal aspects of animal protection.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-ethoxy-2,3-difluoro-4-{4-[(1s,4r)-4-propylcyclohexyl]cyclohex-1-en-1-yl}benzene

EC Number:

603-006-7

Cas Number:

124728-62-7

Molecular formula:

C23H34F2O

IUPAC Name:

1-ethoxy-2,3-difluoro-4-{4-[(1s,4r)-4-propylcyclohexyl]cyclohex-1-en-1-yl}benzene

Test material form:

solid: crystalline

Test animals / tissue source

Species:

other: hen's eggs, fertilized

Strain:

other: White leghorn

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
-Source: LSL Rhein Main Geflügelvermehrungsbetrieb, Dieburg, Germany

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

The tip of a spatula of the test item was applied on the vascular chorioallantoic membrane (CAM). The application volume of the reference material was 0.3 mL.

Duration of treatment / exposure:

Only eggs without injuries of the membrane or other abnormalities were used for testing. After application of the test material, the blood vessels, including the capillary system, and the albumin were examined and scored for irritating effects (haemorrhage, lysis, and coagulation) over a period of 300 sec. The period between treatment and the onset of the reactions was measured in seconds and recorded with stopwatch and protocol.

Number of animals or in vitro replicates:

6 eggs were used

Details on study design:

The irritating potential of the test item was determined in the hen's egg test on the chorioallantoic membrane (HET-CAM). Fertile hen eggs on day 10 of incubation provided the test system. The eggshell was removed around the air cell by means of a dentist drill and the shell with attached outer membrane and inner egg membrane was carefully opened. After the membrane had been moistened with isotonic sodium chloride solution, it was removed to expose the chorioallantoic membrane (CAM) of the fertilized eggs, which lies immediately below. After application of the test material, the blood vessels, including the capillary system, and the albumin were examined and scored for irritating effects (haemorrhage, lysis, and coagulation) over a period of 300 sec. As reference material dodecyl sulfate sodium salt was tested and evaluated to ensure the sensitivity of the system.

Scoring system according to: Lüpke, NP (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd. Chem. Toxic. 23 (2), 287-291.
Cumulative score Assessment of irritancy
0 – 0.9 None
1 – 4.9 Mild
5 - 8.9 Moderate
9 - 21 Severe

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Score: 9.69

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no irritating effects in the HET-CAM test.

Executive summary:

The irritating potential of the test item was determined in the hen's egg test on the chorioallantoic membrane (HET-CAM). This test is routinely used as a screening assay prior to in vivo tests in rabbits for eye irritation. Fertile hen eggs on day 10 of incubation provided the test system. The eggshell was removed around the air cell by means of a dentist drill. The shell with attached outer membrane and inner egg membrane was carefully opened. After the membrane had been moistened with isotonic sodium chloride solution, it was removed to expose the chorioallantoic membrane (CAM) of the fertilized eggs, which lies immediately below. After application of the test material, the blood vessels, including the capillary system, and the albumin were examined and scored for irritating effects (haemorrhage, lysis, and coagulation) over a period of 300 sec. As reference material dodecyl sulfate sodium salt was tested and evaluated to ensure the sensitivity of the system. For the test item a cumulative score of 0 (no irritancy) was obtained, while dodecyl sulfate sodium salt as reference material gave a score of 9.69 (severe irritancy). Under the given experimental conditions, the test item showed no irritating effects in the HET-CAM test.