Hydrocarbons, terpene processing by-products

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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 test item on a reconstructed human corneal epithelium model by quantification of cellular viability by MTT reduction test (Mosmann, T. 1983) and determination of 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

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 %
- Composition of test material (%): Terpineols (4.4 %), alpha pinene (1.1 %), alpha fenchene (0.2 %), camphene (1.0 %), alpha phellandrene (0.5 %), alpha terpinene (2.7 %), cineol 1.4 (20.5 %), d-limonene (9.1 %), l-limonene (9.1 %), beta phellandrene (0.2 %), paracymene (0.7 %), cineol 1.8 (14.6 %), gamma terpinene (3.6%), terpinolene (31.8 %) 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 in darkness

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control: sodium chloride (0.9 % w/v) in sterile water; positive control: 1.5 % (w/w) SDS in sterile water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration: Test material: undiluted; negative control: sodium chloride (0.9 % w/v); positive control: 1.5 % (w/w) SDS solution

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

Number of animals or in vitro replicates:

Test item, negative control and positive control were tested in duplicate

Details on study design:

TEST SYSTEM:
- Cell system used: Human reconstructed corneal epithelium model of 0.5 cm² (5 days of culture), from immortalized human corneal epithelial cells from the cell line HCE (Human Corneal Epithelial cells), cultivated in chemically defined medium
- Source: SKINETHIC Laboratories (Lyon, France)

METHODOLOGY:
- Non-specific MTT reduction: 10 µL of the test item was added to each well of a 12-wells plate containing 2 mL of MTT solution (0.3 mg/mL) and colour of the solution was checked after incubation for 3 h ± 5 min at 37 ± 1 °C, 5 ± 1 % CO2, 95 ± 5 % humidity (CO2 incubator).
- MTT conversion assay: The epithelial units were transferred from the kit into maintenance medium filled wells and pre-incubated at 37 ± 1 °C until the following day. After pre-incubation, 30 µL of test material, in duplicate, were applied to the epithelium model for 10 ± 2 min, 1 h ± 10 min and 3 h ± 30 min at room temperature. Contact timepoint for negative control was 3 h ± 30 min and for positive controls were 10 ± 2 min and 1 h ± 10 min. At the end of each incubation period, each epithelium was rinsed under a jet of PBS+ (10 mL), transferred into a new well containing MTT solution and incubated for 1 h ± 10 min in CO2 incubator. Each epithelium was then transferred into a new 24 wells plate containing isopropanol (1 mL). After agitating for 1 h ± 10 min, each extract (200 μL/well) was transferred into a 96 wells microplate and optical density was recorded at 570 nm with a plates reader (blank = isopropanol). For each treated tissue the viability was expressed as mean percentage of cellular viability relative to the negative control and the exposure time that causes 50% of cell mortality (T50) was determined by linear regression analysis.

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: TERPINOLENE MULTICONSTITUENT did not interact with MTT

Any other information on results incl. tables

Table 7.3.2/2: Optical density readings 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, the T50 value of the test item, TERPINOLENE MULTICONSTITUENT, was between 10 and 60 min therefore it was 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. Contact timepoint for negative control (sodium chloride 0.9 % w/v) was 3 h ± 30 min and 10 ± 2 min and 1 h ± 10 min for positive control (SDS 1.5% w/w). At the end of each incubation period, each epithelium was rinsed, transferred to new well containing MTT solution and incubated for 1 h ± 10 min in CO2 incubator. Each epithelium was then transferred into a new well plate containing isopropanol (1 mL). After agitating for 1 h ± 10 min, the extract (200 μL/well) was transferred into a 96 wells microplate and optical density was recorded at 570 nm with a plates reader. For each treated tissue the viability was expressed as mean percentage of cellular viability relative to the negative control and the exposure time that causes 50% of cell mortality (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, the T50 value of the test item, TERPINOLENE MULTICONSTITUENT, was between 10 and 60 min therefore it was considered as moderately irritant when applied on a human reconstructed corneal epithelium.