[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-05 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 with a minor deviation: difference of viability between negative control tissues replicates was greater than 20%. Considering the results obtained, this deviation was considered as without impact on the conclusion of the 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:

adopted 28 July 2015

Deviations:

yes

Remarks:

difference of viability between negative control tissues replicates was greater than 20%

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

EC Number:

264-935-8

EC Name:

[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

Cas Number:

64519-44-4

Molecular formula:

C15H25NO3

IUPAC Name:

[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

Test material form:

liquid

Details on test material:

Batch No: G17066-2
Substance type: Monoconstituent
CAS-No: 64519-44-4
EINECS-No: 264-935-8
Description: Yellow to pale pink liquid
Test substance storage: Keep in a tightly closed container in a cool and dry place, protected from light.
Date of manufacture: March 2017
Date of expiration: 13/03/2019

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

Test animals / tissue source

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23778] were received on 03 May 2017.

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): 50 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 25 minutes at room temperature
- Post-exposure incubation period: 18 hours at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
The direct interaction of MTT with the test item was checked by adding approximatively 100 mg of the test item, previously applied on a nylon mesh, to 1 mL of solution of MTT at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The colouration potential of the test item in water or isopropanol was checked by adding approximatively 100 mg of the test item to 1 mL of distilled water or 2 mL of isopropanol, respectively.


MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 19 hours and 50 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied on a nylon mesh at an approximate amount of 50 mg, and then applied to the entire surface of 2 living RhCE tissue replicates during 6 hours at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 6 hours at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 18-hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 2 mL of a MTT solution at 1.0 mg/mL for 2 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 2 hours at room temperature in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test

Value:

3.11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 36.9°C and 37.6°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained in water after 1 hour of incubation between 36.9°C and 37.6°C, 5% CO2. A colourless solution was obtained in isopropanol after 3 hours of incubation at room temperature. No significant colouration appeared, therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- The mean percent tissue viability of the RhCE replicates treated with the test substance was 3.11%, versus 24.05% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.
- The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validated the experiment.

Any other information on results incl. tables

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

	Tissue	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Difference of viability %
Negative control	1	0.475	0.471	0.564	83.58	100.00	32.83
		0.477
		0.461
	2	0.668	0.656		116.42
		0.638
		0.663
Positive control	1	0.120	0.130	0.136	23.07	24.05	1.95
		0.134
		0.138
	2	PH	0.141		25.02
		0.142
		0.139
Test item	1	0.018	0.017	0.018	3.02	3.11	0.18
		0.016
		0.017
	2	0.018	0.018		3.19
		0.018
		0.020

#: mean of 3 values (triplicate of the same extract)

OD: optical density

 

Acceptability criteria:

- The difference of viability between the two replicates of the negative control group was 32.83% instead of <20%. Considering the results obtained, this deviation was considered as without impact on the conclusion and the validity of the study.

Applicant's summary and conclusion

Interpretation of results:

other: Category 2 (irritating to eyes) or Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

An in vitro eye irritation test using the Reconstructed Human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

The test item was applied on a nylon mesh, at an approximate amount of 50 mg in order to cover the entire surface of the epidermis and it was subsequently applied on epidermis. The test item was applied to 2 DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and an 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test item was 3.11%, versus 24.05% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).