Essential oil of Cedarwood Texas obtained from the wood of Juniperus mexicana (Cupressaceae) by distillation - Terpenes

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Apr 2017 - 04 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 26, 2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,4a,8,8-tetramethyl-1H,1aH,4H,4aH,5H,6H,7H,8H-cyclopropa[e]naphthalene; 2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-ene

EC Number:

946-670-6

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

2,4a,8,8-tetramethyl-1H,1aH,4H,4aH,5H,6H,7H,8H-cyclopropa[e]naphthalene; 2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-ene

Test material form:

liquid

Remarks:

Almost colourless, very pale yellow liquid

Details on test material:

Name of test material as cited in study report: Cedarwood Texas oil Terpenes 2 (Thujopsene)
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor; batch: B-64543
- Expiration date of the lot/batch: 25 January 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was tested neat.

OTHER SPECIFICS: UVCB

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor; batch: B-64543
- Expiration date of the lot/batch: 25 January 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was tested neat.

OTHER SPECIFICS: UVCB

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:Vitelco, 's Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)).
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples: no

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μl)
- Concentration (if solution): pure

Duration of treatment / exposure:

10 +/- 1 minutes

Duration of post- treatment incubation (in vitro):

120 +/- 10 minutes

Number of animals or in vitro replicates:

triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder). The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C prior to the start of the exposure

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the received corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
three

NEGATIVE CONTROL USED
Yes, untreated

POSITIVE CONTROL USED
Yes, Ethanol

APPLICATION DOSE AND EXPOSURE TIME
Dose: pure 100%
Exposure time: 10 minutes

TREATMENT METHOD: closed chamber

POST-EXPOSURE INCUBATION PERIOD: yes 120 +/- 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 2 (once with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:

The opacity of a cornea was measured by measuring the diminution of light passing through the cornea. The light is measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) is calculated according to:
Opacity= (empirically determined illuminance through a cornea holder, devided by the measured illuminance through a holder with cornea - 0,9894)/0,0251
The change in opacity for each individual cornea (including the negative control) is calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity of each cornea treated with the test item or positive control is calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group is calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

In vitro score ranges used:
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Main (triplicate)

Value:

-1 - 0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

-0.3 - 0.5

Positive controls validity:

valid

Remarks:

45.2 - 61.5

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Negative control Opacity -2.9 – 3.0
Negative control Permeability -0.016 – 0.042
Negative control In vitro Irritancy Score -2.8 – 3.0
Positive control In vitro Irritancy Score 34.7 – 78.2

Applicant's summary and conclusion

Interpretation of results:

other: Not irritating

Remarks:

Based on CLP (1272/2008/EC).

Conclusions:

Based on the results obtained in the presented study, Cedarwood Texas oil distilled - Terpenes (Thujopsene) does not need to be classified for eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The eye hazard potential of Cedarwood Texas oil distilled - Terpenes (Thujopsene) was determined by measuring its ability to induce opacity and increase permeability according to OECDTG 437. The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied as it is (750 μl) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment. In conclusion, since Cedarwood Texas oil distilled - Terpenes (Thujopsene) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).