Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-02-2020 to 14-04-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: May 2018 ; signature: August 2018

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Refrigerated (2-8°C), under inert gas and protected from light.
- Other: colourless

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): ca. 7 weeks old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, 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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)
- Time interval prior to initiating testing: < 24 hours. Eyes were prepared for testing immediately on same day arrival within 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: None. Only eyes free from damage
- Indication of any antibiotics used: None reported.

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): 0.03 mL (or 30 µL of the test item)
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of physiological saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.
Observation period (in vivo):
Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been rinsed with the physiological saline.
Number of animals or in vitro replicates:
Triplicate (n=3)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report. Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, 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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 to 60 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: Yes,.

SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report. Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, 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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 to 60 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: Yes,.

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED : Benzalkonium chloride (5%)

APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.03 mL (or 30 µL of the test item) ; Exposure time: 10 seconds.

OBSERVATION PERIOD: prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL physiological saline. Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes after the eyes had been rinsed with the physiological saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable.

SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 2.
- Mean maximum opacity score: See tables 1 to 2.
- Mean fluorescein retention score at 30 minutes post-treatment: See tables 1 to 2.

DECISION CRITERIA: In accordance with guideline OECD TG 438.
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium, sticking of test item to the cornea). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test item exposure) were determined at each of the above time points.
ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438.
The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.

A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were within the historic control data range and/or identified as GHS Non Classified and GHS Category 1, respectively

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean (n=3)
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
mean (n=3)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
mean (n=3)
Value:
-3.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value %; ICE Class I
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

Any other information on results incl. tables

Table 1. Ocular reactions

Test Item

 

 

Maximal mean score for corneal opacity

0.33

ICE Class I

Mean score of Fluorescein Retention

0.00

ICE Class I

Corneal swelling

-3.9%

ICE Class I

 

 

 

Positive Control Item

 

 

Maximal mean score for corneal opacity

4.00

ICE Class IV

Mean score of Fluorescein Retention

2.83

ICE Class IV

Corneal swelling

23.1%

ICE Class III

 

 

 

Negative Control Item

 

 

Maximal mean score for corneal opacity

0.00

ICE Class I

Mean score of Fluorescein Retention

0.00

ICE Class I

Corneal swelling

0.00%

ICE Class I

 

- Corneal Opacity Scores

Maximum score was 0.5 (n=2 of 3) in the test item group. Maximum score was 0.0 in the negative control. In the positive control group the Maximum score was 4.0. No morphological effects were noted in the test item or negative control item treated eyes. In the positive control group, severe loosening of epithelium was observed in one eye at 75 minutes after the post-treatment rinse.

 

- Fluorescein Retention Scores

Maximum score was 0.0 (n=3 of 3) in the test item group and negative control. In the positive control group the Maximum score was 3.00.

 

Table 2. Individual scoring - test item

End Point

Eye Number

Time (after decontamination)

0 minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

12

0

0

0

0

0

0

13

0

0

0

0.5

0.5

0.5

14

0

0

0

0.5

0.5

0.5

Mean

0.00

0.00

0.00

0.33

0.33

0.33

ICE Class

I

Fluorescein Retention

12

0.5

0.5

 

 

 

 

13

0.0

0.0

 

 

 

 

14

0.0

0.0

 

 

 

 

Mean difference (30 - 0 min)

 

0.00

 

 

 

 

ICE Class

I

Corneal Thickness

3A

0.60

0.60

0.60

0.58

0.58

0.58

6A

0.62

0.62

0.62

0.62

0.60

0.60

8A

0.60

0.60

0.60

0.59

0.57

0.57

Mean

0.61

0.61

0.61

0.60

0.58

0.58

Mean Corneal Swelling (%)

 

0.0

0.0

-1.7

-3.9

-3.9

ICE Class

I

ICE Classes Combined:

3 x I

Classification:

Category 1

 

The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item was considered to be non-irritant.
Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. The method is also able to identify substances not requiring UN GHS classification.After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL test item. The three positive control eyes were treated in a similar way 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). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. For the test item the mean corneal opacity was 0.33 (ICE Class I). The mean fluorescein retention was 0.0 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class V with maximal corneal swelling -3.9%. No other morphological effect was observed. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV across cornel opacity and fluorescein retention categories and Class III in mean corneal thickness) signifying that the test system performed adequately. Under the conditions of this study, the test item was considered to be non-irritant.