Reaction mass of α-3,3-trimethyl-(αS,1R)-cyclohexanemethanol and α-3,3-trimethyl-(αR,1S)-cyclohexanemethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June - 28 July 2016

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 in viability between two replicates for the test item and positive control was slightly greater than 20%. Considering the results obtained, this minor 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 in viability between two replicates for the test item and positive control was slightly greater than 20%

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Test material

Test material information

Constituent 1

Reference substance name:

(αS,1R)-α-3,3-trimethyl-cyclohexanemethanol

Cas Number:

49817-42-7

Molecular formula:

C10H20O

IUPAC Name:

(αS,1R)-α-3,3-trimethyl-cyclohexanemethanol

Constituent 2

Reference substance name:

(αR,1S)-α-3,3-trimethyl-cyclohexanemethanol

Cas Number:

49817-52-9

Molecular formula:

C10H20O

IUPAC Name:

(αR,1S)-α-3,3-trimethyl-cyclohexanemethanol

impurity 1

Reference substance name:

(1R-trans)-2,6,6-trimethyl-cycloheptanol

Cas Number:

49817-55-2

Molecular formula:

C10H20O

IUPAC Name:

(1R-trans)-2,6,6-trimethyl-cycloheptanol

impurity 2

Reference substance name:

(1S-trans)-2,6,6-trimethyl-cycloheptanol

Cas Number:

49817-44-9

Molecular formula:

C10H20O

IUPAC Name:

(1S-trans)-2,6,6-trimethyl-cycloheptanol

impurity 3

Reference substance name:

(R*,R*)-α,3,3-trimethyl-cyclohexanemethanol

Molecular formula:

C10H20O

IUPAC Name:

(R*,R*)-α,3,3-trimethyl-cyclohexanemethanol

impurity 4

Reference substance name:

(S*,S*)-α-3,3-trimethyl-cyclohexanemethanol

Molecular formula:

C10H20O

IUPAC Name:

(S*,S*)-α-3,3-trimethyl-cyclohexanemethanol

impurity 5

Reference substance name:

(1R-cis)-2,6,6-trimethyl-cycloheptanol

Cas Number:

49817-43-8

Molecular formula:

C10H20O

IUPAC Name:

(1R-cis)-2,6,6-trimethyl-cycloheptanol

impurity 6

Reference substance name:

(1S-cis)-2,6,6-trimethyl-cycloheptanol

Cas Number:

49817-56-3

Molecular formula:

C10H20O

IUPAC Name:

(1S-cis)-2,6,6-trimethyl-cycloheptanol

impurity 7

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 179585
Purity: 71.4% (sum of the two main constituents)
Name of test material (as cited in study report): α-3,3-TRIMETHYLCYCLOHEXANEMETHANOL MULTICONSTITUENT
Physical state: colourless to very pale yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 01 March 2018

Test animals / tissue source

Species:

human

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. 23721] were received on 26 July 2016.

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 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 2 hours and 2 minutes 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 50 µL of the test item to 1 mL of MTT solution 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 50 µL 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 20 hours and 10 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, as supplied, at the dose of 50 µL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes 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 30 minutes 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 for comparable colour with the negative control. This rinsing step was followed by a 12-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 2-hour and 10-minute 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 300 µL of a MTT solution at 1.0 mg/mL for 3 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 17 hours and 05 minutes at 6±3°C 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 at 570 nm was measured in triplicate samples of formazan extracts. The measurement of OD was performed 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:

18.66

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 37.1°C and 37.8°C, 5% CO2. Therefore, there is 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 37.4°C and 37.9°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
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 18.66%, versus 22.84% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

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	0.954	0.946	0.994	95.17	100.00	9.66
		0.941
		0.944
	2	1.005	1.042		104.83
		1.079
		1.042
Positive control	1	0.361	0.365	0.227	36.72	22.84	27.77
		0.342
		0.394
	2	0.084	0.089		8.95
		0.091
		0.093
Test item	1	0.334	0.323	0.186	32.49	18.66	27.67
		0.312
		0.323
	2	0.050	0.048		4.83
		0.047
		0.049

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

OD: optical density

 

Note

- The difference in viability between two replicates was 27.67% for the test item and 27.77% for the positive control instead of less than 20%. Considering the results obtained, these deviations were considered as without impact on the conclusion of the study.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test substance was identified as potentially requiring classification and labelling according to UN GHS and CLP Regulation (Category 2 or Category 1). The results of a corrosivity study (OECD 437) have to be taken into account to confirm the classification of the substance.

Executive summary:

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

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour and 2-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

In the preliminary tests, the test substance 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 substance was 18.66%, versus 22.84% in the positive control (Methyl acetate).

Therefore, the test substance was identified as potentially requiring classification and labelling according to UN GHS and CLP Regulation (Category 2 or Category 1). The results of a corrosivity study (OECD 437) have to be taken into account to confirm the classification of the substance.