Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-16 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with generally accepted scientific standards and described in sufficient details

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline available

Deviations:

not applicable

Principles of method if other than guideline:

Evaluation of the primary ocular irritation after application of the test item on a reconstructed human corneal epithelium model by quantification of cellular viability (MTT reduction test - Mosmann T. 1983) and determination of the exposure time that causes 50% of cell mortality (T50).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

EC Number:

938-945-4

Molecular formula:

Not applicable

IUPAC Name:

Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): terpinolene multiconstituent
- Physical state: yellow liquid
- Analytical purity: 66.9 % (sum of the three main constituents)
- Composition of test material (%): terpinolene (31.8 %), 1,4-cineole (20.5 %), 1,8-cineole (14.6 %), d-and l-limonene (18.2 %), terpineols (4.4 %), gamma terpinene (3.6%), alpha terpinene (2.7 %), alpha pinene (1.1 %), camphene (1.0 %), paracymene (0.7 %), alpha phellandrene (0.5 %), alpha fenchene (0.2 %), beta phellandrene (0.2 %) and others (0.5 %)
- Lot/batch No.: 123238
- Purity test date: 17 October 2011
- Date of receipt: 16 April 2012
- Expiration date of the lot/batch: 29 September 2012
- Storage condition of test material: stored at 6 ± 3°C protected from light

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

In vitro assay

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration: test material: undiluted; negative control: sodium chloride at 0.9 % w/v in water; positive control: sodium dodecyl sulfate at 1.5 % (w/w) in water

Duration of treatment / exposure:

- Test item: 10 ± 2 min, 1 h ± 10 min and 3 h ± 30 min
- Positive control: 10 ± 2 min and 1 h ± 10 min
- Negative control: 3 h ± 30 min

Observation period (in vivo):

Post-exposure period: approximately 2 h

Details on study design:

TEST SYSTEM:
- Cell system used: Human reconstructed corneal epithelium model of 0.5 cm² (5 days of culture). When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human corneal epithelial cells from the cell line HCE (Human Corneal Epithelial cells) reconstruct a corneal epithelial tissue (mucosa), devoided of stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.
- Source: SkinEthic Laboratories (Lyon, France)

CONTROLS AND REPLICATES:
- Negative control: sodium chloride at 0.9% w/v in sterile water
- Positive control: sodium dodecyl sulfate at 1.5% w/w in sterile water
The test item and the negative and positive controls were tested in duplicate.

METHODOLOGY:
- Treatment
Upon arrival the epithelia were transferred to new maintenance medium, 500 µL per well in 24-well plates, and incubated at 37°C ± 1°C in a CO2 incubator (5 ± 1% CO2, 95 ± 5% humidity). The medium was renewed 24 hours later (500 µL per well) and 30 µL of the test material or 30 µL of each control were topically applied on the tissue surface.
The tissues treated with the test item were incubated in the CO2 incubator for 10 ± 2 min, 1 hour ± 10 min or 3 hours ± 30 min. The tissues treated with the positive contol were incubated for 10 ± 2 min or 1 hour ± 10 min. The tissues exposed to the negative control were incubated for 3 hours ± 30 min. Two epithelia were used for each treatment and exposure period.
- Viability measurement using the MTT assay
(MTT reduction into formazan by cellular enzymes)
At the end of the treatment period, the epithelia were rinsed with 10 mL of PBS+ (Phosphate Buffered Saline) and transferred into wells containing 500 µL of a MTT solution at 0.5 mg/mL [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The tissues were incubated for 1 hour ± 10 min in the CO2 incubator.
Each epithelium was then removed from the MTT solution, blotted on absorbent paper and transferred to isopropanol (1 mL per well - 24-well plates). After 1 hour ± 10 min of gentle stirring protected from light, 200 µL aliquots per well were transferred to 96-well plates before measuring optical density (OD) at 570 nm with isopropanol as the blank, using a microtiter plate reader.
- Expression of viability
Viability was expressed as % relative to the negative control: [test item mean OD/negative control mean OD] x 100. The exposure time that reduced cell viability to 50% of negative control level (T50) was determined by linear regression analysis.
- Non-specific MTT reduction
Before being assayed, the test item was tested for its potential interaction with the MTT reagent. 10 µL of the test item was added to 2 mL of a MTT solution at 0.3 mg/mL in each well of a 12-well plate. After mixing and incubation for 3 hours ± 5 min at 37°C ± 1°C (no coloration means no interaction between the test item and the MTT reagent).

Results and discussion

In vivo

Results

Irritation parameter:

other: T50

Basis:

mean

Time point:

other: 42.88 min

Reversibility:

other: not applicable

Irritant / corrosive response data:

Negative control:
- Mean OD: 1.152

T50 values:
- Positive control: 33.49 min
- Test material: 42.88 min

- See table 7.3.2/2 for more data

Other effects:

- Non-specific MTT reduction: the test item did not interact with MTT

Any other information on results incl. tables

Table 7.3.2/2: Optical density results and % viability

 

	Contact Timepoint (min.)	O.D. 1	O.D. 2	Mean O.D.	% viability
Negative control	180	1.179	1.125	1.152	100%
Positive control	10	1.093	1.000	1.047	91%
	60	0.054	0.030	0.042	4%
Test item	10	0.951	1.118	1.035	90%
	60	0.317	0.359	0.338	29%
	180	0.150	0.117	0.134	12%

O.D.: Optical density

Applicant's summary and conclusion

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: See table 7.3.2/1

Conclusions:

Under the test conditions, T50 value of the test item, TERPINOLENE MULTICONSTITUENT, was found between 10 and 60 min (42.88 min). It was therefore considered as moderately irritant when applied on a human reconstructed corneal epithelium.

Executive summary:

In an in vitro eye irritation study performed in compliance with GLP, 30 µL of the test item, TERPINOLENE MULTICONSTITUENT was applied to the human reconstructed corneal epithelium model (in duplicate) for 10 ± 2 min, 1 h ± 10 min and 3 h ± 30 min at room temperature. Exposure time for negative control (sodium chloride at 0.9 % w/v) was 3 h ± 30 min. Exposure times for positive control (SDS 1.5% w/w) were 10 ± 2 min and 1 hour ± 10 min. At the end of the treatment period, the epithelia were rinsed, transferred to well plates containing a MTT solution for 1 h ± 10 min. Each epithelium was then removed from the MTT solution and transferred to isopropanol (1 mL per well). After stirring for 1 h ± 10 min, aliquots (200 μL/well) were transferred to 96-well plates and optical density was measured at 570 nm with a plate reader. For each treatment the viability was expressed as mean percentage of cellular viability relative to the negative control and the exposure time that reduced cell viability to 50% of negative control level (T50) was determined by linear regression analysis.

T50 value for the test item was 42.88 min. Optical density value for the negative control was 1.152 and T50 value for positive control was 33.49 min and thus confirmed the validity of the test.

Under the test conditions, T50 value of the test item, TERPINOLENE MULTICONSTITUENT, was between 10 and 60 min. It was therefore considered as moderately irritant when applied on a human reconstructed corneal epithelium.