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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:
23 August - 22 September, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

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

Materials and methods

Test guideline
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)
Version / remarks:
26 July, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: Leuco Sulfur Red 14
Appearance: light red solid
CAS No: 68585-53-5
EC No: 271-559-8

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source:
TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: 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 (19.4 ºC to 20.3 ºC) 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).
- indication of any existing defects or lesions in ocular tissue samples:
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:
TEST MATERIAL
- Amount applied: 30 mg, 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.

Duration of treatment / exposure:
10 s
Duration of post- treatment incubation (in vitro):
30 min
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:
SELECTION AND PREPARATION OF ISOLATED EYES
See "Details on test animals or tissues and environmental conditions"

EQUILIBRATION AND BASELINE RECORDINGS
appropriate eyey for the test,were acclimated for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods

NUMBER OF REPLICATES

3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.

NEGATIVE CONTROL USED

Sodium chloride, NaCl (9 g/L saline)

POSITIVE CONTROL USED

Imidazole

APPLICATION DOSE AND EXPOSURE TIME

NaCl: 30 µL/eye
Imidazole: 30 mg/eye

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
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.


METHODS FOR MEASURED ENDPOINTS:
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 .
- Corneal opacity:
- Damage to epithelium based on fluorescein retention:
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
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.
- Macroscopic morphological damage to the surface:
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.


SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
The negative controls showed the expected results. The experiment was considered to be valid.
Positive controls valid:
yes
Remarks:
The positive controls showed the expected results. The experiment was considered to be valid.
Remarks on result:
no indication of irritation
Remarks:
The overall ICE class was 1xI, 2xII; thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made"

Any other information on results incl. tables

Table 5: Summary of the test item results 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

3%

I

Mean maximum corneal swelling at up to 240 min

4%

I

Mean maximum corneal opacity

1.0

II

Mean fluorescein retention

1.0

II

Other Observations

None

Overall ICE Class1

1xI, 2xII

 

Table 6: Summary of the positive control results 

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, UN GHS Classification: Category 1.

Table 7: Summary of the negative control results 

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:
other: in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.
Conclusions:
In an eye irritation assay (isolated chicken eye) according to OECD guideline 438, no prediction could be made.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The study was performed according to the OECD guideline 438. 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. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

 The test item and Imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or 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 (9 g/L). After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with ca. 20 mL saline solution at ambient temperature. Positive and negative controls showed the expected results. The experiment was considered to be valid. In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with Leuco Sulfur Red 14, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 2xII.

According to the overall in vitro evaluation criteria classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.