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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:
23 August - 28 September, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
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:
26 July, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine
EC Number:
290-904-3
EC Name:
Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine
Cas Number:
90268-98-7
Molecular formula:
Molecular formula is not available
IUPAC Name:
Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine
Test material form:
solid: particulate/powder
Details on test material:
Test item: Yellow 22
Appearance: ocher clay, solid
CAS No. 90268-98-7
EC No. 290-904-3
Storage: room temperature

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal
(19.4ºC to 20.3ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 5 heads/box).
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied in its original form, no formulation was required. The test item and the positive control were finely ground before application.
The test item and positive control applied in an amount of 0.03 g/eye.
Duration of treatment / exposure:
The exposure period was 10 seconds.
Number of animals or in vitro replicates:
3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.
Details on study design:
isolated chicken eye test (ICET)
Removal of test item: The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.
The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 minutes of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance .

Tool used to assess score: hand-slit lamp/biomicroscope/fluorescein
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
the test item is categorized as "NO CATEGORY"; ICE classes: 3xI

Any other information on results incl. tables

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.

Positive and negative controls showed the expected results. The experiment was considered to be valid

 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

1%

I

Mean maximum corneal swelling at up to 240 min

1%

I

Mean maximum corneal opacity

0.5

I

Mean fluorescein retention

0.5

I

Other Observations

None

Overall ICE Class1

3xI

 

 

Positive Control: Imidazole

 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

25%

III

Mean maximum corneal swelling at up to 240 min

33%

IV

Mean maximum corneal opacity

4.0

IV

Mean fluorescein retention

3.0

IV

Other Observations

Cornea opacity score 4 was observed in two eyes at
30 minutes after the post-treatment rinse.

Overall ICE Class1

3xIV

 

The positive control Imidazole was classed as corrosive/severely irritating,UNGHS Classification: Category 1

Negative Control: NaCl (9 g/L saline)

 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

2%

I

Mean maximum corneal swelling at up to 240 min

3%

I

Mean maximum corneal opacity

0.5

I

Mean fluorescein retention

0.0

I

Other Observations

None

Overall ICE Class1

3xI

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model, no ocular corrosion or severe irritation potential was observed.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. Imidazole was used as positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µL saline solution. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, the test item is categorized as “No Category”.