Resinoid of Commiphora erythraea (Burseraceae) obtained from the gum by ethanol extraction

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 438 without any deviation.

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 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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on 17 June 2015 / signed on 24 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Commiphora erythraea (Burseraceae) obtained from the gum by ethanol extraction

EC Number:

944-533-5

Molecular formula:

not applicable (UVCB)

IUPAC Name:

Resinoid of Commiphora erythraea (Burseraceae) obtained from the gum by ethanol extraction

Test material form:

solid

Remarks:

Brown red paste

Details on test material:

- Storage condition of test material: Room temperature, in the dark

Specific details on test material used for the study:

- Purity test date: 15 October 2015
- Expiration date of the lot/batch: 23 July 2016

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads, were supplied by Baileys Turkeys Ltd., Cheshire, UK where they were killed for human consumption were used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): Spring chickens (Gallus Gallus e.g. Ross 308 Broiler) weighed 3kg and were approximately 56 days old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads (9:55am- 11 January 2016) and placing the eyes in the superfusion chamber (11:10am- 11 January 2016) following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. The temperature of the chambers was at 32 ±1.5 °C.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

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): 30 µL of the test item was applied, as supplied, to the cornea
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline

Number of animals or in vitro replicates:

Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are =< 0.5.
- Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
- Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.
- Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
- After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 2, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. 1.
- Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein
- Pitting, loosening (sloughing), roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Value:

14.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 1.0, corresponding to the ICE class II;
- mean score of fluorescein retention: 0.5, corresponding to the ICE class I;
- maximal mean corneal swelling: 14.69% corresponding to the ICE class III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as "No Category", as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Any other information on results incl. tables

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment with the test item

 

End Point	Eye Number	Time (after decontamination)
		0 minutes	30 minutes	75 minutes	120 minutes	180 minutes	240 minutes
Corneal Opacity	3B	0.5	0.5	0.5	1	1	1
	6B	0.5	1	1	1	1	1
	8B	0	0.5	0.5	0.5	1	1
Mean		0.3	0.7	0.7	0.8	1.0	1.0
ICE Class		II
Fluorescein Retention	3B		0.5
	6B		0.5
	8B		0.5
Mean			0.5
ICE Class		I
Corneal Thickness	3B	0.73	0.70	0.84	0.79	0.80	0.82
	6B	0.68	0.68	0.74	0.68	0.72	0.78
	8B	0.70	0.68	0.84	0.80	0.78	0.78
Mean		0.70	0.69	0.81	0.76	0.77	0.79
Mean Corneal Swelling (%)			-2.37	14.69	7.58	9.00	12.80
ICE Class		III
ICE Classes Combined:		II, I, III
Classification:		No prediction of eye irritation can be made

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the test conditions, no prediction can be made according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item was applied, as supplied, at the dose of 30 µL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.0, corresponding to the ICE class II;

- mean score of fluorescein retention: 0.5, corresponding to the ICE class I;

- maximal mean corneal swelling: 14.69% corresponding to the ICE class III.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

 

Under the test conditions, no prediction can be made according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP).