β,4-dimethylcyclohex-3-ene-1-propan-1-al

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

β,4-dimethylcyclohex-3-ene-1-propan-1-al

EC Number:

229-846-0

EC Name:

β,4-dimethylcyclohex-3-ene-1-propan-1-al

Cas Number:

6784-13-0

Molecular formula:

C11H18O

IUPAC Name:

3-(4-methylcyclohex-3-en-1-yl)butanal

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Storage, temperature, and transport conditions of ocular tissue: Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old and 2.55 kg) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to the test site at ambient temperature at the earliest convenience. The heads were received at test site and were processed within 2 hours of collection.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL per cornea.

POSITIVE CONTROL
- Amount applied: 30 µL per cornea.

NEGATIVE CONTROL
- Amount applied: 30 µL per cornea.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 replicates for the positive and treatment group each and one for the negative group

Details on study design:

SOLUBILITY CHECKING
The solubility of the test item in physiological saline was tested prior to the experiment (approximately 30 μL test material in 1 mL physiological saline). The test item dissolved in physiological saline.

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
Eyes were examined with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No corneal thickness changes were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

ADMINISTRATION OF TEST AND CONTROL ITEMS
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. 30 μL of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea. The positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

REMOVAL OF TEST SUBSTANCE
After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature to remove all residual test material without damaging the cornea.

OBSERVATION PERIOD
-Corneal thickness and corneal opacity: Pre- treatment and at approximately (±5 minutes) 30, 75, 120, 180 and 240 minutes after the post-treatment rinse
-Fluorescein retention: Pre- treatment and at approximately (±5 minutes) 30 minutes after the post-treatment rinse

METHODS FOR MEASURED ENDPOINTS:
- Corneal swelling: An optical pachymeter on a slit lamp microscope
- Corneal opacity: slit lamp microscope
- Fluorescein retention: slit lamp microscope

SCORING SYSTEM:
- Mean corneal swelling (%):
corneal swelling (t) = (cornea thickness (t) – cornea thickness at t=0) / (cornea thickness at t=0) x 100
Mean corneal swelling (t) = (1st eye corneal swelling (t)+ 2nd eye corneal swelling (t) +3rd eye corneal swelling (t)) / 3

- Mean maximum opacity score:
Δcornea opacity (t) = cornea opacity (t) – cornea opacity at t=0
Mean Δcornea opacity (max) = (1st eye cornea opacity max(30min to 240min)+ 2nd cornea opacity eye max(30min to 240min) + 3rd eye cornea opacity max(30min to 240min)) / 3

- Mean fluorescein retention score at 30 minutes post-treatment:
Δfluorescein retention(t) = fluorescein retention (t) – fluorescein retention at t=0
Mean Δfluorescein retention = (1st eye fluorescein retention (30min) + 2nd eye fluorescein retention (30min) + 3rd eye fluorescein retention (30min)) / 3

For the calculation of maximum corneal swelling, small negative numbers for swelling following application (0 to -5%) are counted as zero (scored as class I). Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

DECISION CRITERIA: The mean maximum corneal swelling up to 75 or 240 min, the mean maximum corneal opacity and the mean fluorescein retention change were assigned ICE classes which are used for classification according to UN GHS (2019) criteria. (see Tables 1 t/m 5)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean maximum corneal swelling at up to 75 min

Run / experiment:

Mean of 3 replicates (%)

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean maximum corneal swelling: 0%

Positive controls validity:

valid

Remarks:

Mean maximum corneal swelling: 13.6%

Remarks on result:

other: ICE CLASS I

Results 2

Irritation parameter:

other: Mean maximum corneal swelling at up to 240 min

Run / experiment:

Mean of 3 replicates (%)

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean maximum corneal swelling: 0%

Positive controls validity:

valid

Remarks:

Mean maximum corneal swelling: 27.2%

Remarks on result:

other: ICE CLASS I

Results 3

Irritation parameter:

other: Mean maximum corneal opacity change

Run / experiment:

mean of 3 replicates

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean maximum corneal opacity change: 0

Positive controls validity:

valid

Remarks:

Mean maximum corneal opacity change: 4

Remarks on result:

other: ICE CLASS I

Results 4

Irritation parameter:

other: Mean fluorescein retention change

Run / experiment:

Mean of 3 replicates

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean fluorescein retention change: 0

Positive controls validity:

valid

Remarks:

Mean fluorescein retention change: 3

Remarks on result:

other: ICE CLASS I

Other effects / acceptance of results:

Severe loosening of epithelium was observed in all three eyes at 30 minutes after the post treatment rinse for the positive control. No other morphological effect was observed in the study.

For detailed results see "attached full study report".
See table 6 for interpretation of the obtained results according to the UN GHS classification scheme

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical control data range. This study was considered to be valid. See "attached full study report" for the historical control data.

Any other information on results incl. tables

Table 6. SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the posttreatment rinse:	True

 

Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False

 

Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

Applicant's summary and conclusion

Interpretation of results:

other: Not eye irritant in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

Based on the results of an in vitro eye irritation assay in isolated chicken eyes performed according to OECD guideline 438 and GLP principles, the substance is a non irritant.

Executive summary:

An Isolated Chicken Eye Test (ICET) was performed with the substance according to OECD guideline 438 and in accordance with GLP principles. Thirty µL of the substance was applied to corneas (n=3). After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. No significant corneal swelling change (mean = 1.2) was observed during the four-hour observation period on test item treated eyes. No cornea opacity and no fluorescein retention change were observed on the test item treated eyes. No other corneal effect was observed. The negative control and positive control results were within the historical data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Based on the results, the  Corneal opacity, Corneal swelling and Fluorescein retention endpoints were assigned ICE CLASS I. Therefore, the substance is considered as non-irritant.