Cyclohexanone, 2-methyl-4-(1,1,2-trimethylpropyl)-, cis-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 November 2010 - 9 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD guideline 438 (7 September 2009)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.48 (ICE test 8 December 2010)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of (2R,4R)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone and (2S,4S)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone

EC Number:

938-347-3

Cas Number:

28068-91-9

Molecular formula:

C13H24O

IUPAC Name:

Reaction mass of (2R,4R)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone and (2S,4S)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone

Details on test material:

- Name of test material (as cited in study report): Iriswood
- Substance type: pure active substance
- Physical state: undercooled liquid

Test animals / tissue source

Species:

other: Spring chicken

Details on test animals or tissues and environmental conditions:

The eyes collected from chickens obtained from a slaughterhouse. Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with isotonic saline.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

Amount / concentration applied:

30 µl of pure test substance or control was put onto enucleated chicken eyes during 10s.

Duration of treatment / exposure:

The test substance was applied for 10s and then rinsed from the eye with 20 ml isotonic saline incubation period was 10±1 min at 32±1.5°C.

Observation period (in vivo):

Corneal opacity, swelling and morphological effects were evaluated at 30, 75, 120, 180 and 240 minutes post-dosing. Fluorescein retention was determined at pretreatment and 30 minutes post-dosing.

Number of animals or in vitro replicates:

3 eyes per dose group (except negative control: 1 eye).

Details on study design:

The eyelids were excised and dissected from the skull. The eyeball was pulled from the orbit and placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The cornea of the enucleated eye was positioned vertically by a steel clamp and placed in a superfusion apparatus in such a way that the entire cornea was supplied with the isotonic saline drip.
The eyes were then examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was measured at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. Individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
The approved eyes were incubated between 60 and 76 minutes prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline. The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
Following the zero reference measurements, the eye was removed from the superfusion apparatus, placed in a horizontal position. 30 μL of the test substance was applied to the cornea for 10 seconds, such that the entire surface of the cornea is evenly covered with the test item. The test substance was applied and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature. The eye was subsequently returned to the superfusion apparatus in the original upright position for further measurements.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

corneal swelling 

Run / experiment:

1

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Assessment eye corrosion/severe irritation of an eye exposed to physiological saline (negative control), 10% acetic acid (positive control) and the test substance.

		time / min
	End-point	0	30	75	120	180	240
Negative control	Corneal opacity	0	0	0	0	0	0
	Fluorescein retention	0.5	0.5
	Corneal swelling / %	-	4	4	4	4	4
Positive control	Corneal opacity	0	2.0	2.0	2.0	2.0	2.0
	Fluorescein retention	0.2	3.0
	Corneal swelling / %	-	22	46	33	35	27
Test substance	Corneal opacity	0.0	0.0	0.0	0.5	0.5	0.5
	Fluorescein retention	0.2	0.0
	Corneal swelling / %	-	2	3	9	9	8

ICE classification of the negative control, positive control and the test substance.

	End-point	ICE
Negative control	Corneal opacity	I (=1)
	Fluorescein retention	I (=1)
	Corneal swelling	I (=1)
Positive control	Corneal opacity	III (=3)
	Fluorescein retention	IV (=4)
	Corneal swelling	IV (=4)
Test substance	Corneal opacity	I (=1)
	Fluorescein retention	I (=1)
	Corneal swelling	II (=2)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test substance must not be classified as causing “irreversible effects on the eye” under the conditions of the in vitro test.

Executive summary:

The eye irritancy of Iriswood was assessed according to the Isolated Chicken Eye Test as described in OECD method 438. 30ml test substance was applied to 3 enucleated chicken eyes during 10s. The eyes were subsequently rinsed with 10ml of physiological saline. Three other eyes were treated with a positive control and one eye with a negative control. The damage by the test substance was assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The ocular reactions observed in the eyes treated with the test substance were slightly to moderate. Based on the results of the test, the test substance does not need to be classified as causing “irreversible effects on the eye”.