α-methylcyclohexanepropanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-11-16 to 2015-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July, 2015).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo CRS GmbH, 64380 Rossdorf, Germany

Test material

Test material information

Constituent 1

Reference substance name:

α-methylcyclohexanepropanol

EC Number:

234-091-5

EC Name:

α-methylcyclohexanepropanol

Cas Number:

10528-67-3

Molecular formula:

C10H20O

IUPAC Name:

4-cyclohexylbutan-2-ol

Test animals / tissue source

Species:

human

Strain:

other: Reconstructed human Cornea-like Epithelium (RhCE)

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
RhCE tissue was used.
Cells used for the tissue production were screened for biological contamination and determined to be free of any contamination.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Duration of treatment / exposure:

30 min for test item and controls

Duration of post- treatment incubation (in vitro):

122 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number
- EpiOcular Kit (Lot number: 21581) Kits MatTek Corporation (Ashland, MA 01721, USA)
- Doses of test chemical and control substances used: 50 μL
- Duration and temperature of exposure (30 min, 37 ± 1.5 °C), post-exposure immersion (12 ± 2 minutes, room temperature) and post-exposure incubation (122 minutes, 37 ± 1.5 °C) periods
- Description of any modifications to the test procedure: No modifications were done.
- Number of tissue replicates used per test chemical and controls: 2
- Description of the method used to quantify MTT formazan: OD measurement, no data on wavelength etc provided
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The mean OD value of the blank control wells (ODBlk) for each experiment were calculated. The ODBlk from each OD value of the same experiment (blank corrected values) were subtracted. The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated. The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed. The corrected OD value of the negative control corresponds to 100% viability. Corrected negative control OD = Negative Control OD - ODBfk = 100% Viability
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
- Positive and negative control means and acceptance ranges based on historical data:
negative control OD: > 0.8 and < 2.5; mean relative viability of the positive control: below 60% of the negative control viability
- Acceptable variability between tissue replicates for positive and negative controls: < 20%
- Acceptable variability between tissue replicates for the test chemical: < 20%

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability [%]

Run / experiment:

mean

Value:

39.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

32.0 %

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No damages described.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Results:

 

Dose Group	Mean Absorbance* Tissue 1 and 2 minus Mean Blank	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]**
Negative control	1.339	1.307	102.5	5.0	100.0
	1.274		97.5
Positive control	0.442	0.418	33.8	3.6	32.0
	0.395		30.2
Test item	0.531	0.514	40.6	2.6	39.3
	0.497		38.0

* Mean of three replicate wells after blank correction

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

 

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 39.3 % (threshold for irritancy:≤60 %), consequently the test item was irritant to eye.

 

Concerning acceptance criteria:

-The negative control OD is >0.8 and < 2.5 (1.274 and 1.339).

-The mean relative viability of the positive control is below 60 % of the negative control viability (32.0 %).

-The difference of viability between the two relating tissues of a single item is < 20 % (values between 2.6 % to 5.0 %) in the same run (for positive and negative control tissues and tissues of single test items).

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 or 2 based on GHS criteria

Conclusions:

The test substance was determined to be an eye irritant in the in vitro Reconstructed human Cornea-like Epithelium.

Executive summary:

The in vitro Reconstructed human Cornea-like Epithelium study according to OECD 492 was performed to assess the irritation potential of the test substance. Independent duplicate tissues of EpiOcular were exposed to either the test item, the negative control (deionised water) or the positive control (Methyl acetate) for 30 min. 50 μL of the test item were dispensed directly onto duplicate EpiOcular tissue surface. After exposure of the tissues to the test substance the mean tissue viability decreased to 39.3 %. This value is below the threshold for irritation, which is defined to be ≤ 60 %. Therefore, the test substance was considered to be eye irritating or cause serious damage to the eye.