4-cyclohexyl-4-methylpentan-2-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.06.2016 - 06.10.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-cyclohexyl-4-methylpentan-2-one

EC Number:

225-562-6

EC Name:

4-cyclohexyl-4-methylpentan-2-one

Cas Number:

4927-39-3

Molecular formula:

C12H22O

IUPAC Name:

4-cyclohexyl-4-methylpentan-2-one

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- EpiOcularTM tissue: normal, human-derived epidermal keratinocytes cultured to form a stratified squamous epithelium
- Surface: 0.6 cm²
- Shipment: at 2 - 8 °C on medium-supplemented agarose gels
- Equilibration step: 15 minutes at room temperature
- Inspection: each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Pre-incubation: standard culture conditions for 1 hour and after refreshing the Assay Medium at standard culture conditions overnight (21 h in the 1st experiment, 18 h in the 2nd experiment).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Volume: 50 μL
- Concentration: 83.3 μL/cm2

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2

Details on study design:

Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular Kit Lot No.: 23714 (1st experiment) and 23737 (2nd experiment)
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Washing: extensively rinsing the tissues with Ca++Mg++-free DPBS
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm (OD570), Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1. No reference wavelength measurement was used.
- MTT assay: incubation with 0.3 mL of MTT solution for 180 minutes at standard culture conditions, after incubation with MTT the inserts were incubated with isopropanol at 2-8°C overnight after which MTT was extracted for 2-3 hours at room temperature
- Data evaluation: the following was calculated: mean of the blank control wells (ODBlk), ODBlk from each OD value of the same experiment (blank corrected values), mean of the two aliquots for each tissue (= corrected test item OD), mean of the two relating tissues for each control and test item (= corrected mean OD) (for further calculations only the corrected mean negative control OD value was needed), corrected OD value of the negative control corresponding to 100% viability (corrected negative control OD = Negative Control OD - ODBlk = 100% Viability)
- Description of evaluation criteria: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is non-irritant and if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
A single test composed of at least 2 tissue replicates should be sufficient for a test chemical when the result is unequivocal. In cases of borderline results, such as non- concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests (according to OECD 492).
- Historical data positive control: Mean Viability: 32.3%; Rel. Standard Deviation: 11.1%; Range of Viabilities: 6.90% - 43.4%; Mean Absorption: 0.566; Rel. Standard Deviation: 0.283; Range of Absorbance: 0.107- 0.943
- Historical data negative control: Mean Absorption: 1.65; Rel. Standard Deviation: 0.295; Range of Absorbance: 1.27 – 2.16
- Acceptability of the Assay: The results are acceptable if (1) The negative control OD is > 0.8 and < 2.5, (2) The mean relative viability of the positive control is below 50% of the negative control viability. (3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

First experiment

Value:

61.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: borderline result

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Second experiment

Value:

79.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The acceptance criteria were met.

Any other information on results incl. tables

Results after treatment for 30 minutes

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean Absorbance* (Tissue 1/2)	Mean Absorbance * Tissue 1 and 2	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Rel. Absorbance [% of Negative Control]**
First experiment
Blank	0.039	0.040	0.039	0.000
Negative Control	2.160	2.155	2.158	2.118	2.066	102.5	5.1	100.0
	2.049	2.056	2.053	2.013		97.5
Positive Control	0.579	0.574	0.577	0.538	0.721	26.0	17.7	34.9
	0.955	0.931	0.943	0.904		43.7
Test Item	1.377	1.321	1.349	1.310	1.272	63.4	3.7	61.5
	1.275	1.271	1.273	1.233		59.7
Second experiment
Blank	0.039	0.039	0.039	0.000
Negative Control	2.110	2.057	2.084	2.045	2.023	101.1	2.2	100.0
	2.027	2.052	2.040	2.001		98.9
Positive Control	0.943	0.921	0.932	0.893	0.878	44.2	1.6	43.4
	0.908	0.892	0.900	0.862		42.6
Test Item	1.624	1.571	1.597	1.558	1.607	77.0	4.9	79.5
	1.732	1.659	1.695	1.657		81.9

* Mean of two replicate wells after blank correction


** Relative absorbance [rounded values]: 100 x (absorbance test item / positive control) / absorbance negative control

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

The test item is not eye irritant.

Executive summary:

In the current study the eye irritation potential of the test item was assessed by means of the Human Cornea Model Test. The test was according to OECD 492 and GLP.

Important to mention is that the first experiment did not give a clear result, because of this a second confirmation experiment was performed.

In the pre-tests, the test item did not reduce MTT and the intrinsic colour of the substance was not intensive and did not color water or isopropanol. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

For the main test, 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied to tissue for 30 minutes and the test was performed in duplicate.

The absorbance values after treatment with the negative control were well within the required acceptability criterion showing the quality of the tissues, while treatment with the positive control induced a decrease below 50% compared to the negative control ensuring the validity of the test system.

The test was also valid as the difference in viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Treatment with the test item did not indicate irritating effects. In the first experiment the relative mean absorption value corresponding to the viability of the tissues was 61.5%. This result lies within the borderline range of 60±5%, thus, a a second test was performed. In the second experiment the mean adsorption was 79.5%, giving a clear result.

In conclusion, in this study and under the experimental conditions reported, the test item is not eye irritant.