Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-04-2017

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:

26 July, 2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

EC Number:

946-913-6

Molecular formula:

Not applicable for UVCB Substance

IUPAC Name:

Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

Test material form:

liquid

Remarks:

pale yellow liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

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
- Source and lot/batch No.of test material: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

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

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Test System
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Age at study initiation: young cattle
- Other info: the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

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 of either the negative control, positive control or undiluted test item was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

Duration of post- treatment incubation (in vitro):

Corneas were incubated for 120 +/- 10 minutes at 32 +/- 1°C with cMEM.

Number of animals or in vitro replicates:

Triplicate

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 (Earle’s Minimum Essential Medium 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) of BASF 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 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.
QUALITY CHECK OF THE ISOLATED CORNEAS:
-Opacity determinations were performed on each of the 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: 3
NEGATIVE CONTROL USED:
- A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
POSITIVE CONTROL USED:
- Ethanol, Batch K47177483, Identification number RS532, Purity >99.9% Stable under storage conditions until 31 October 2020
APPLICATION DOSE AND EXPOSURE TIME:
- Undiluted, 10 +/- 1 minutes at 32 +/- 1°C
TREATMENT METHOD:
- The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- POST-EXPOSURE INCUBATION: yes; 120 +/- 10 minutes at 32 +/- 1°C.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
2) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value) Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. 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) are: ≤ 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 experiment

Value:

ca. -0.7 - -0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
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 61 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 (SD 1.07, n=72)
-permeability -0.016-0.042 (SD 0.01, n=65)
-IVIS -2.8-3.0 (SD 1.17, n=66)
Positive control:
-IVIS 34.7-78.2 (SD 12.64, n=47)

Any other information on results incl. tables

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity (1)	Mean Permeability (1)	Mean In vitro Irritation Score (1,2)
Negative control	-0.5	0.003	-0.4
Positive control (Ethanol)	19.6	2.737	60.7
Copaiba balsam oil	-0.5	0.009	-0.4

(1) Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

(2) In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:

other: not classified

Remarks:

based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Copaiba balsam oil induced an IVIS ≤ 3. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The eye hazard potential of Copaiba balsam oil was evaluated according to OECD guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 μl of the undiluted Copaiba balsam oilfor 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Copaiba balsam oil did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of -0.4 after 10 minutes of treatment.

In conclusion, since Copaiba balsam oil induced an IVIS ≤ 3, and the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).