Resinoid of Myroxylon balsamum var. pereirae (Fabaceae) obtained from the exudate 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:

October 2017 - February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 438 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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:

dated 26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

dated 08 December 2010

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Myroxylon balsamum var. pereirae (Fabaceae) obtained from the exudate by ethanol extraction

EC Number:

947-269-9

IUPAC Name:

Resinoid of Myroxylon balsamum var. pereirae (Fabaceae) obtained from the exudate by ethanol extraction

Test material form:

liquid

Details on test material:

Manufacturing date: 14/04/2017
Expiry date: 8/03/2018 (prolonged to 01/08/2019)

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 11 Ocotber 2017 at 8:25 am.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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. The eyes were enucleated at Phycher on 11 October 2017 at 9:55 am.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None

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): The test item was applied, as supplied, at the dose of 30 μL, to 3 enucleated chicken eyes, during 10 seconds.

Duration of treatment / exposure:

10 seconds

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. 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 were at a controlled temperature between 32.9 °C and 33.5 °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.
- Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 48 and 60 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.

NUMBER OF REPLICATES
- 1, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus and placed in a horizontal position. The test item was applied, as supplied, at the dose of 30 μL, to 3 enucleated chicken eyes, during 10 seconds.

REMOVAL OF TEST SUBSTANCE
- After exposure, the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. 1. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
The mean percentage of corneal swelling for all tested 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 the test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested 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
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: The mean fluorescein retention value for all tested 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
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

DECISION 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 the test item.
Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean of three runs

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean of three runs

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

mean of three runs

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OCULAR REACTIONS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.2, corresponding to ICE class I;
- mean score of fluorescein retention: 1.7, corresponding to ICE class III;
- maximal mean corneal swelling: 4%, corresponding to ICE class I.
The combination of the three endpoints for the test item was 2 x I, 1 x III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. 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

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	4	0.00	0.00	0.00	0.00	0.00	0.50
	5	0.00	0.00	0.00	0.00	0.00	0.00
	6	0.00	0.00	0.00	0.00	0.00	0.00
Mean		0.00	0.00	0.00	0.00	0.00	0.20
ICE class		I
Fluorescein retention	4	0.50	1.00	-	-	-	-
	5	0.50	2.00	-	-	-	-
	6	0.50	2.00	-	-	-	-
Mean		0.50	1.70	-	-	-	-
ICE class		III
Corneal thickness	4	0.60	0.60	0.61	0.61	0.61	0.61
	5	0.58	0.60	0.62	0.62	0.64	0.64
	6	0.56	0.56	0.56	0.56	0.56	0.56
Corneal swelling (%)	4	-	0.00	2.00	2.00	2.00	2.00
	5	-	3.00	7.00	7.00	10.00	10.00
	6	-	0.00	0.00	0.00	0.00	0.00
Mean		-	1.00	3.00	3.00	4.00	4.00
ICE class		I
Combination of the 3 Endpoints		2 x I, 1 x III
CLASSIFICATION		No prediction can be made

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained lead to the category “no prediction can be made”, as defined by the OECD guideline No. 438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Executive summary:

An ex vivo 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 twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item 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: 0.2, corresponding to ICE class I;

- mean score of fluorescein retention: 1.7, corresponding to ICE class III;

- maximal mean corneal swelling: 4%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 2 x I, 1 x III.

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.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained lead to the category “no prediction can be made”, as defined by the OECD guideline No. 438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.