Amyris balsamifera, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-09-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amyris balsamifera, ext.

EC Number:

291-076-6

EC Name:

Amyris balsamifera, ext.

Cas Number:

90320-49-3

Molecular formula:

Not applicable due to the UVCB nature of the substance.

IUPAC Name:

Essential oil of Amyris obtained from the wood of Amyris balsamifera (Rutaceae) trees by steam distillation.

Test material form:

liquid: viscous

Remarks:

Pale yellow slightly viscous liquid

Details on test material:

Name of test material as cited in study report: Amyris oil
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test item :207778/A
- Source and lot/batch No.of test material: L4275185
- Expiration date of the lot/batch: 31 October 2017 (retest date)

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

Test animals / tissue source

Species:

other: Bovine

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 positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

After exposure, corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3

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 containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C. The corneas were incubated for the minimum of 1 hour at 32°C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. 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: 3

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME:
-750 µl of either the positive control, the negative control or the test item, Corneas were incubated in a horizontal position for 10 minutes at 32°C
TREATMENT METHOD: [closed chamber / open chamber]
POST-INCUBATION PERIOD: -

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- Post-exposure incubation: Corneas were incubated for 120 minutes at 32°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity value is calculated (measured with the device OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others: After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
-The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN
GHS Category 1) and test items not requiring classification for eye irritation or serious eye
damage (UN GHS No Category)
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
-Acceptability of the assay: 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.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

26.3

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.6

Positive controls validity:

valid

Remarks:

69.5

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

other: not classified

Remarks:

based on GHS and CLP

Conclusions:

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.2 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test, in accordance with UN GHS, and the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

In this Bovine Corneal Opacity and Permeability test (BCOP test), the eye hazard potential of Amyris oil was tested. 750 µl of the testing material was tested through topical application directly on top of the corneas for 10 minutes. The negative and positive controls for opacity and permeability were within the laboratory historical range indicating 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 vitro irritancy score of 1.2 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test.