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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 14th, 2016.
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:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): 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): 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.
- Time interval prior to initiating testing: The heads were collected on 14 November 2016 at 8:15 am. The eyes were enucleated at Phycher on 14 November 2016 at 9:35 am.
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.

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
Number of animals or in vitro replicates:
3 replicates per group (test item, positive control, negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage. 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: Once all eyes had been examined and approved, the eyes were incubated between 45 and 65 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: 3

NEGATIVE CONTROL USED: 30 µL physiological saline – Dutscher Batch No. 3012316.

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED: 30 mg sodium hydroxide – Sigma, Batch No. MKBP7805V.

APPLICATION DOSE AND EXPOSURE TIME: 30 mg test item, exposure 10 seconds.

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

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the test item was rinsed from the eye with 20 mL of physiological saline at ambient temperature. As test item remained on the cornea despite the rinsing, 2 additional rinses were performed with 10 mL of physiological saline. A cotton swab moistened with physiological saline was used to gently remove all the test item from the cornea. A last rinse was performed with 10 mL of physiological saline.
- Indicate any deviation from test procedure in the Guideline: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 test 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.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention value for all test 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.
- Swelling: measured with optical pachymeter on a slit-lamp microscope (HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I); slit-width setting: the slit-width was set at 9½, equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. The value was determined from corneal thickness measurements. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Macroscopic morphological damage to the surface: qualitative assessment. The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.
- Others (e.g, histopathology): no.

SCORING SYSTEM:
- Mean corneal swelling (%) was calculated as follows: [(corneal thickness at time t - corneal thickness at time 0)/corneal thickness at time 0] x 100.
- Mean maximum opacity score:
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

DECISION CRITERIA: as indicated in the TG.

Results and discussion

Results of ex vivo / in vitro studyopen allclose all
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE class III
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE class II
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE class I
Irritation parameter:
morphological effects
Run / experiment:
mean
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no. No morphological effects were noted, whatever the examination time.
- Other observations: the test item required additional rinsing to be removed from the cornea.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

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: no.

Any other information on results incl. tables

Table 1. Test item results (30 mg), 14/11/2016.

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

13

0.5

1

1

1

1

1

14

0

0.5

0.5

0.5

1

1

15

0

0.5

0.5

0.5

0.5

0.5

Mean

0.2

0.7

0.7

0.7

0.8

0.8

ICE class

II

Fluorescein retention

13

0.5

2

-

-

-

-

14

0.5

2

-

-

-

-

15

0.5

2

-

-

-

-

Mean

0.5

2.0

-

-

-

-

ICE class

III

Corneal thickness

13

0.62

0.63

0.63

0.63

0.63

0.63

14

0.63

0.65

0.65

0.65

0.65

0.65

15

0.60

0.60

0.60

0.60

0.60

0.60

Corneal swelling (%)

13

-

2

2

2

2

2

14

-

3

3

3

3

3

15

-

0

0

0

0

0

Mean

-

2

2

2

2

2

ICE class

I

Classification

1 x III, 1 x II, 1 x I

No prediction can be made

No morphological effects were noted, whatever the examination time.

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
EU criteria
Conclusions:
The combinations of the 3 endpoints for the test item were 1 x III, 1 x II, 1 x I. Therefore, no prediction can be made.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30 µL of physiological saline (negative control). Three eyeballs were used in each group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints for the test item were 1 x III, 1 x II, 1 x I.