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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:
key study
Study period:
March 27, 2017
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

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 have been collected on 27 March 2017 at 8: 20 am.The eyes were enucleated at Phycher on 27 March 2017 at 10:00 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 of test item
Duration of treatment / exposure:
10 second
Duration of post- treatment incubation (in vitro):
No post-treatment incubation is performed.
Number of animals or in vitro replicates:
3
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 32.0°C and 32.5°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 using sodium fluorescein. 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. (see table appendix No.4 on "Any other information on results incl. tables")
Once all eyes had been examined and approved, the eyes were incubated between 45 and 56 minutes to equilibrate them to the test system prior to dosing.

EQUILIBRATION AND BASELINE RECORDINGS:
Eyes were incubated between 45 and 56 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 (one eye)

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED
Sodium hydroxide – Sigma, Batch No. MKBP7805V - 30 mg (three eyes)

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied for 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 after 10 seconds of observation with 20 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: NO

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It 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 (see table 4)
- Damage to epithelium based on fluorescein retention: 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 (Table No.5)
- Swelling: optical pachymeter on a slit-lamp microscope ((HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I). The slit-width was set at 9 1/2 equalling 0.095 mm. The mean percentage of corneal swelling for all test 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 each test item (see table 3).
- Macroscopic morphological damage to the surface: 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.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling: It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*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 ris 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: Decision criteria was used as indicated in the TG.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean
Value:
ca. 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE class I
Irritation parameter:
morphological effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No effects
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: NO, No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY:Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:YES, the combination of the three endpoints for the negative control, physiological saline, was 3xI classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV,classified as “Corrosive/Severe Irritant ”

Any other information on results incl. tables

Appendix No. 4: Selected eyes for the performance of the ICE test

Chamber

Fluorescein

retention

Corneal

opacity

Morphological

effects

Corneal thickness (e)

1

0.5

0

N.t.R.

0.58

2

0.5

0

N.t.R.

0.59

3

0.5

0

N.t.R.

0.54

4

0.5

0

N.t.R.

0.60

5

0.5

0

N.t.R.

0.60

6

0.5

0

N.t.R.

0.59

7

0.5

0

N.t.R.

0.62

8

0.5

0

N.t.R.

0.60

9

0.5

0

N.t.R.

0.59

10

0.5

0

N.t.R.

0.59

Table 9: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Test item

Endpoint measured

Eye No.

0

30

75

120

180

240

 

 

Corneal opacity

4

0

0

0

0

0

0.5

5

0

0

0

0

0

0.5

6

0

0

0

0

0

0.5

Mean

 

0.0

0.0

0.0

0.0

0.0

0.5

ICE class

 

 

 

 

I

 

 

 

Fluorescein retention

4

0.5

0.5

 

 

 

 

5

0.5

0.5

 

 

 

 

6

0.5

0.5

 

 

 

 

Mean

 

0.5

0.5

 

 

 

 

ICE class

 

 

I

 

 

 

 

 

 

 

Corneal thickness

4

0.60

0.62

0.62

0.62

0.62

0.62

5

0.60

0.61

0.61

0.61

0.61

0.61

6

0.59

0.59

0.59

0.59

0.59

0.59

 

Corneal swelling (%)

4

-

3

3

3

3

3

5

-

2

2

2

2

2

6

-

0

0

0

0

0

Mean

 

 

2

2

2

2

2

ICE class

 

 

 

 

I

 

 

Combination of the 3 Endpoints

3 x I

CLASSIFICATION

No category

Note: No morphological effects were noted, whatever the examination time.

Table 8: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Positive control

Endpoint measured

Eye No.

0

30

75

120

180

240

 

 

Corneal opacity

1

0

4

4

4

4

4

2

0

4

4

4

4

4

3

0

4

4

4

4

4

Mean

 

0.0

4.0

4.0

4.0

4.0

4.0

ICE class

 

 

 

 

IV

 

 

 

Fluorescein retention

1

0.5

3

 

 

 

 

2

0.5

3

 

 

 

 

3

0.5

3

 

 

 

 

Mean

 

0.5

3.0

 

 

 

 

ICE class

 

 

IV

 

 

 

 

 

 

 

Corneal thickness

1

0.58

-

-

-

-

-

2

0.59

 -

-

-

-

-

3

0.54

-

-

-

-

-

 

Corneal swelling (%)

1

-

(-)

(-)

(-)

(-)

(-)

2

-

(-)

(-)

(-)

(-)

(-)

3

-

(-)

(-)

(-)

(-)

(-)

Mean

 

-

-

-

-

-

-

ICE class

 

 

 

 

IV

 

 

Combination of the 3 Endpoints

3 x IV

CLASSIFICATION

Category 1 : Corrosive/Severe irritant

Note:

(-): evaluation of corneal swelling not possible (Corneal opacity = 4 at each examination time, leading to a marked refraction

of the light preventing from the evaluation of the corneal swelling with the biomicroscope).

Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Negative control

Endpoint measured

Eye No.

0

30

75

120

180

240

Corneal opacity

10

0.0

0.0

0.0

0.0

0.0

0.0

ICE class

 

 

 

 

I

 

 

Fluorescein retention

10

0.5

0.5

-

-

-

-

ICE class

 

 

I

 

 

 

 

Corneal thickness

10

0.59

0.60

0.60

0.60

0.60

0.60

Corneal swelling (%)

10

-

2

2

2

2

2

ICE class

 

 

 

 

I

 

 

Combination of the 3 Endpoints

3 x I

CLASSIFICATION

No Category

Note: No morphological effects were noted, whatever the examination time.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The test item isoborneol was determined to not cause severe damage or irrritation for eyes in the ICE test.
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 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control 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 CLP Regulation EC no. 1272/2008 the test item isoborneol does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combinations of the 3 endpoints for the test item were 3x I.