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

Endpoint summary

Description of key information

The in-vitro skin irritation test (OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method) showed that the test substance should be considered as an irritant to skin.

The in-vitro eye irritation test (OECD guideline 438: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants) showed that the test substance does not have irreversible effects on the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin 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:

other: Study conducted by GLP accredited laboratory. Method according to OECD guideline.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Type of coverage:

open

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

10µl undiluted test material. The same amount is applied to the positive and negative control.

Duration of treatment / exposure:

15 minutes at room temperature.

Details on study design:

Prior to the analysis, the test substance was checked for possible direct reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) (MTT).

10 µl of the test substance was added to 2.2 ml MTT solution and incubated for 3h at 37°C, 5% CO2. A negative control of distilled water was included.
Three tissues per test were treated with the test material. Three tissues were treated with the negative control and three tissues were exposed to the positive control (5% aqueous solution of sodium dodecylsulphate-SDS CAS 151-21-3). The tissue samples were exposed to the test substance / controls for 15 minutes. After the exposure, the tissues were rinsed with phosphate buffered saline (PBS) and incubated for 42h at 37°C, 5% CO2.
 
After the incubation, the tissues were subsequently incubated for another 3h in the presence of 2.2ml MTT. The formed formazan was extracted with 0.5 ml isopropanol for 2h under agitation in the dark. The amount of Formazan was determined spectrophotometrically (Optical Density - OD) at 570 nm in duplicate. The cell viability was measured against the mean of the negative control.

Irritation / corrosion parameter:

other: other: Viability

Value:

14.6

Remarks on result:

other:

Remarks:

Basis: mean. Max. score: 100.0. (migrated information)

Irritant / corrosive response data:

The test substance was visually checked for possible direct MTT reduction causing a colour change. No interaction with MTT was observed.
Details of the data and the mean of the spectroscopic measurements can be found in Table 1. Table 2 presents the mean relative tissue viability obtained after 15 minutes treatment with the test substance being the mean of the absorption of the test substance divided by the negative control mean for each application. Since the mean relative tissue viability of the test substance was below 50% after an exposure duration of 15 minutes, the test substance is considered to be irritant.

Table 1  Absorptions (OD₅₇₀) in the in vitro skin irritation test

	15-minute exposure
	1	2	3	Mean
Negative control	0.548	0.529	0.507	0.528
Test substance	0.066	0.101	0.064	0.077
Positive control	0.104	0.071	0.076	0.084

Table 2 Mean tissue viability as % of negative control in the in vitro skin irritation

Exposure	15-minutes	SD
	%
Negative control	100	3.8
Test substance	18	3.9
Positive control	10	3.4

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

The test is complies with the acceptability criteria and the test substance is considered an irritant in the in vitro skin irritation test under the experimental conditions used.

Executive summary:

An in vitro skin irritation test was conducted on a human skin model according to the OECD guideline 439. The basis of the test is that the cell viability was determined by using the MTT reduction assay. The test substance was applied undiluted on top of the skin tissue for 15 minutes. After the exposure, the tissue was incubated with MTT and the formazan that was produced from MTT was spectroscopically determined at 570 nm. The relative mean tissue viability of the test substance expressed as the absorption of the test substance against the negative control showed that the test substance has a relative mean tissue viability of ≤50% compared to the negative control. The test substance is therefore considered an irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

other: OECD guideline 438 (7 September 2009)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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.

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.

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

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

Irritation parameter:

corneal swelling 

Run / experiment:

1

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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)

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”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:

Study conducted on substance by a GLP accredited laboratory. Method according to OECD guideline.

Justification for selection of eye irritation endpoint:

Study conducted on substance by a GLP accredited laboratory. Method according to OECD guideline.

Effects on skin irritation/corrosion: irritating