Oils, cinnamon

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-08-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Essential oil of Cinnamon bark obtained from the bark of Cinnamomum zeylanicum, Lauraceae by distillation

EC Number:

943-043-9

Cas Number:

8015-91-6

IUPAC Name:

Essential oil of Cinnamon bark obtained from the bark of Cinnamomum zeylanicum, Lauraceae by distillation

Test material form:

liquid

Remarks:

Pale yellow to dark yellow or brownish yellow color and powerful diffusive warm-spicy sweat and tenacious odour.

Details on test material:

Identification: Cinnamon Bark Oil (CBO)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: fridge, dry, protected from heat and light
- Container: aluminium flask

OTHER SPECIFICS
- Form: liquid
- Colour: pale yellow to dark yellow or brownish yellow
- Odour: powerful diffusive warm-spicy sweet and tenacious

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: chicken heads obtained from Etablissement Brun (address: 33820 Etauliers, France)
- Number of animals: multiple
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old, 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads have been collected on 16 August 2021 at 8:11 am. The eyes were enucleated at Laboratoire ICARE –Site de Martillac on 16 August 2021 at 9:36 am.
- Selection and preparation of corneas: Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The enucleated eye was mounted in a stainless-steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.6°C and 31.8°C.
- Quality check of the isolated corneas: After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The cornea was rinsed twice with 10 mL of physiological saline at ambiant temperature. Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless-steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.6°C and 31.8°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated in the superfusion apparatus between 45 and 65 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero-reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After 10 seconds of exposure the surface of the cornea was rinsed with 20 mL physiological saline

METHODS FOR MEASURED ENDPOINTS:
Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. was used for the measurements.
- Corneal opacity: The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Slith-width was set at 9½, equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Macroscopic morphological damage to the surface: include “pitting” of corneal epithelial cells, loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology):

DECISION CRITERIA: Assessment of the general IN VITRO eye irritancy and regulatory UN GHS
classification according to OECD No. 438 (2018).

VALIDATION CRITERIA: Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test item.
The study is considered valid, if:
-the number of eyes meets the requirement of the relevant OECD guideline,
-the negative control item results No Category,
-the positive control item results Category 1 classification.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean at all observation time points

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score at 30 min

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

corneal swelling 

Run / experiment:

maximal mean % for all observation time points

Value:

7

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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, valid positive and negative controls

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean corneal opacity 0, mean Fluorescein
retention 0.5, Mean corneal swelling 0; 3x ICE class I)
- Acceptance criteria met for positive control: yes (mean corneal opacity up to 3.0, mean Fluorescein retention 3.0, Mean corneal swelling up to 47%; 3x ICE class IV)

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the substance is not predicted as causing serious eye damage (Cat. 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

Executive summary:

In an isolated chicken eye test, performed according to the 438 OECD Guideline under GLP conditions, test item was tested to evaluate the eye hazard potential of the substance. 30 μL of the test item was applied to the cornea of each of 3 chicken enucleated eyes and after 10 seconds the corneas were rinsed with physiological saline. After the exposure, toxic effects were measured by qualitative assessment of opacity (0.7, ICE class II), fluorescein retention (1.3, ICE class II), and corneal swelling (7% ICE class II). Valid positive (3x ICE class IV) and negative (3x ICE class I) controls were included. According to the overall in vitro irritancy criteria, the ICE classes combined for test item lead to result ‘no prediction can be made'.