1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-02-2015 to 17-04-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2014; signature: May 2014

Test material

Test material information

Constituent 1

Reference substance name:

1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

EC Number:

807-612-4

Cas Number:

1393645-32-3

Molecular formula:

C14H22O

IUPAC Name:

1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: At approximately 4°C, in the dark under nitrogen
- Other: clear colourless

Test animals / tissue source

Species:

other: chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): ca. 7 weeks , 1.5 to 2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Extraction time and placing eyes in the superfusion chamber following enucleation was minimized (typically within 2 hours). Intact heads were stored at ambient temperature in perspex containers, humidified by towels moistened with isotonic saline during transport. For use on the same day. All eyes have to fall within the acceptance criteria used in the ICE guideline.
- Time interval prior to initiating testing: Same day (< 24 hours)
- indication of any existing defects or lesions in ocular tissue samples: No. Eyes that had a high baseline fluorescein staining score (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
- Indication of any antibiotics used: No.

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): 0.03 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.

Observation period (in vivo):

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

Triplicate (n=3) for test item and reference item ; duplicate (n=2) for negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report.

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: sodium chloride solution 0.9% w/v

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED : Benzalkonium chloride (5%)

APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.03 ml ; Exposure time: 10 seconds.

OBSERVATION PERIOD: prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL isotonic saline. Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable. Samples were taken for possible histopathology where relevant.

SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 3.
- Mean maximum opacity score: See tables 1 to 3.
- Mean fluorescein retention score at 30 minutes post-treatment: See tables 1 to 3.

DECISION CRITERIA: In accordance with guideline OECD TG 438.
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test item exposure) were determined at each of the above time points.
ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438.
The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean (n=3)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean (n=3)

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

mean (n=3)

Value:

>= 4.24 - <= 6.74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency test item results are reported in the full study report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Any other information on results incl. tables

Table 1. Ocular reactions

	Reaction	ICE Prediction
Test Item
Maximal mean score for corneal opacity	0.00	ICE Class I
Mean score of Fluorescein Retention	0.50	ICE Class I
Maximal corneal swelling	6.74%	ICE Class II
Positive Control Item
Maximal mean score for corneal opacity	3.00	ICE Class IV
Mean score of Fluorescein Retention	2.67	ICE Class IV
Maximal corneal swelling	71.41%	ICE Class IV
Negative Control Item
Maximal mean score for corneal opacity	0.25	ICE Class I
Mean score of Fluorescein Retention	0.00	ICE Class 1
Maximal corneal swelling	10.40%	ICE Class II

 

- Corneal Opacity Scores

No opacity or cloudiness of the cornea was noted in any replicate. Severe corneal opacity was noted in all positive control treated eyes. Slight opacity of the corneal was noted in one replicate the negative control treated eyes. No morphological effects were noted in the test item or negative control item treated eyes. Severe morphological effects (sloughing) were seen in all positive control eyes.

- Fluorescein Retention Scores

Slight fluorescein adhering was noted in all the test item treated eyes. Large areas of dense fluorescein staining over most of the cornea were noted in two positive control treated eyes. One positive control eye showed a dense layer of fluorescein staining in small areas of the cornea. No fluorescein retention was noted in the negative control treated eyes.

 

Table 2. Individual scoring - test item

End Point	Eye Number	Time (after decontamination)
		0 minutes	30 minutes	75 minutes	120 minutes	180 minutes	240 minutes
Corneal Opacity	1A	0	0	0	0	0	0
	2A	0	0	0	0	0	0
	3A	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE Class		I
Fluorescein Retention	1A		0.5
	2A		0.5
	3A		0.5
Mean			0.5
ICE Class		I
Corneal Thickness (micro-meters)	1A	269	272	307	276	279	291
	2A	283	300	301	310	306	268
	3A	249	263	246	269	264	267
Mean		267.00	278.33	284.67	285.00	283.00	275.33
Mean Corneal Swelling (%)			4.24	6.62	6.74	5.99	3.12
ICE Class		II
ICE Classes Combined:		I, I, II
Classification:		UN GHS - No category

 

The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. Also to identify substances not requiring UN GHS classification. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes remained untreated for control purposes. The mean corneal opacity was 0.0 (ICE Class I). The mean fluorescein retention was 0.5 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class II with maximal corneal swelling 6.74%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class 2xI, 1xII) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV) across all categories signifying that the test system performed adequately. Under the conditions of this study, the test item is considered to not be irritating to the eye.

The applicant recalculated the scoring in accordance with the OECD TG 438 guideline whereby 3xI or 2xI, 1xII or 2xII, 1xI results in a UN GHS classification - no category prediction. Rather than a "non-ocular corrosive / severe irritant" conclusion indicated in previous editions of the OECD TG 438 guideline. The test item responses were within the range of the concurrent negative controls with the exception of Fluorescein Retention, which was consistently low level (score = 0.5) between all replicates. No replicate gave a positive indication of eye irritation. All acceptability criteria were met within the assay.