Fir, Abies balsamea, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

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 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fir, Abies balsamea, ext.

EC Number:

285-364-0

EC Name:

Fir, Abies balsamea, ext.

Cas Number:

85085-34-3

IUPAC Name:

Fir Balsam Oil

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoided contact with iron.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: No dilution performed. Test item applied as-is
- Final preparation of a solid: not applicable/test item is a liquid

Test animals / tissue source

Species:

chicken

Strain:

other: chicken: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Commercial abattoir of chickens for human consumption
- Number of animals: Several (or not relevant)
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were transported at ambient temperature wrapped with tissue paper moistened with saline.
- Time interval prior to initiating testing: Max 2 hours
- indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: Information is not available

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL for test item and both controls

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

One eye was treated with physiological saline (negative control), three eyes with the test item and another three eyes with powdered 5% (w/v) Benzalkonium chloride solution (positive control).

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids on the heads were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the Haag-Streit BP 900® slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One eye was treated with the negative control. Three eyes were treated with the test item and another three eyes with the positive control.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% w/v NaCl)

POSITIVE CONTROL USED
Benzalkonium chloride solution, 50 % in water diluted to obtain a 5% (w/v) solution

APPLICATION DOSE AND EXPOSURE TIME
30 µL was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly for 10 seconds

OBSERVATION PERIOD
Prior to treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (fluorescein retention is determined only prior to treatment and 30 minutes after test chemical exposure)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.
- Indicate any deviation from test procedure in the Guideline: No deviation

EVALUATION
Corneal thickness or swelling determination: Corneal swelling is determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope with the following settings: Slit length/full length/position 8 and Slit width/Fully open/Setting 9.5
Corneal opacity determination: Corneal opacity is scored using the area of the cornea that is most densely opacified with the following settings for the slit lamp microscope: Slit length/full length/position 8 and Slit width/Fully open/Position on indefinite
Fluorescein retention determination: The fluorescein retention will be measured on two occasions, baseline (t=0) and 30 minutes after the post-treatment rinse. the setting for the slip lamp microscope is identical to the opacity assessment, except with the green light filter


SCORING SYSTEM:
- Mean corneal swelling (%) and ICE Class
0-5 I (No Swelling)
>5 to 12 II (Slight Swelling)
: :
>32 IV (Severe Swelling)

- Mean maximum opacity score and ICE Class
0.0 - 0.5 I (No opacity)
0.6 - 1.5 II (Slight opacity)
1.6 - 2.5 III (Moderate opacity)
2.6 - 4.0 IV (Severe or total opacity)

- Mean fluorescein retention score at 30 min post-treatment and ICE class
0.0 - 0.5 I (No fluorescein retention)
0.6 - 1.5 II (Slight fluorescein retention)
1.6 - 2.5 III (Moderate fluorescein retention)
2.6 - 4.0 IV (Severe fluorescein retention)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

See 'Remarks on results'

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Overall ICE Class 3xI, hence, the negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Positive controls validity:

valid

Remarks:

Overall ICE Class : 1xIII 2xIV, hence, the positive control 5% (w/v) Benzalkonium chloride solution was classified as severely irritating, UN GHS Classification: Category 1.

Remarks on result:

no indication of irritation

Remarks:

Overall ICE Class of test item: 3xI, hence, the test item is non-irritant, UN GHS Classification: No Category. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Other effects / acceptance of results:

Morphological effects: In the positive control group, severe loosening of epithelium was observed in one eye at 120 minutes and in one eye at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study.

Any other information on results incl. tables

RESULTS:

Test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.50	I
Mean fluorescein retention	0.17	I
Other Observations	None
Overall ICE Class	3xI

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	7.5%	II
Mean maximum corneal swelling at up to 240 min	23.4%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Severe loosening of epithelium was observed in one eyes at 120 minutes and in one eyes at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

Negative Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditons of this study, Fir Needle Oil Canadian, was found to have an overall ICE Classification of 3xI. Therefore, based on this in vitro eye irritation in the isolated chicken eyes test with Fir Needle Oil Canadian (Fir, Abies Balsamea, ext), the test item is non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 µL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

No corneal swelling was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on the three eyes. No significant fluorescein retention change (severity 0.5) was noted on one eye and no fluorescein retention change was noted on two eyes. No other corneal effect was observed.

Based on this in vitro eye irritation in the isolated chicken eyes test, Fir Needle Oil Canadian is non-irritant, UN GHS Classification: No Category.