Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-07-06 to 2017-07-21
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 guidelineopen allclose all
Qualifier:
according to
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:
yes
Remarks:
The eyes were incubated between 55 and 70 minutes instead of between 45 and 60 minutes, as initially scheduled. This deviation is considered as without impact on the conclusion of the study.
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
yes
Remarks:
he eyes were incubated between 55 and 70 minutes instead of between 45 and 60 minutes, as initially scheduled. This deviation is considered as without impact on the conclusion of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals / tissue source

Species:
other: Eyes of chickens
Details on test animals or tissues and environmental conditions:
- Source: slaughterhouse (Etablissement Brun, 33820 Etauliers, France), chicken killed for human consumption have been used for this assay.
- Number of animals: 7 eyes
- Characteristics of donor animals (e.g. age, sex, weight): spring chickens traditionally processed by a
poultry slaughterhouse (i.e., 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):
* transport time : 1h35
* transport temperature : ambient temperature
* transport conditions : in plastic boxes humidified with towels moistened with physiological saline.

- Time interval prior to initiating testing:
1h35 for the enucleation,
55 and 70 minutes to equilibrate them to the test system prior to dosing, once all eyes had been
examined and approved

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 mg
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
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 was temperature controlled between 31.6°C and 32.4°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
Selected eyes were incubated between 55 and 70 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
- Test item : 3 replicates
- Concurrent negative control: 1 replicate
- Concurrent positive control: 3 replicates

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
sodium hydroxide

APPLICATION DOSE AND EXPOSURE TIME
30 mg of test item and positive control (powder) for 10 seconds evenly covering the surface of the
cornea.
30 μL of negative control for 10 seconds evenly covering the surface of the cornea.

OBSERVATION PERIOD
240 minutes +/- 5 min (observation post-treatment)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: eyes were rinsed four times with 10 mL of physiological saline


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. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
- Corneal opacity: calculated by using the area of the cornea that was most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: calculated for the 30-minute observation time point only
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
[(corneal thickness at time t - corneal thicknes at time =0)/(corneal thickness at time t=0)] x 100
The mean percentage of corneal swelling for all test eyes was calculated for all observation time points.
- 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.

SCORING SYSTEM:
- Mean corneal swelling (%): scoring system as indicated in the TG
- Mean maximum opacity score: scoring system as indicated in the TG was used.
- Mean fluorescein retention score at 30 minutes post-treatment: scoring system as indicated in the TG was used.

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
ca. 0.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class II
Irritation parameter:
fluorescein retention score
Value:
ca. 2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class III
Irritation parameter:
percent corneal swelling
Value:
ca. 2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class I
Other effects / acceptance of results:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, 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, Sodium Hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The test was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
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 test item s 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.
Executive summary:

The aim of the study was 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 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed four times 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 experimental protocol was

established in accordance with the O.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48 – Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

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

- maximal mean score of corneal opacity: 0.7, corresponding to ICE class II;

- mean score of fluorescein retention: 2.0, corresponding to ICE class III;

- maximal mean corneal swelling: 2%, corresponding to ICE class I.

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

The combination of the three endpoints for the positive control, Sodium Hydroxide, 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 2 x I, 1 x II. Therefore, the negative control is classified as “No Category”, as expected.

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 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.