Reaction products of Sodium 1-amino-4-bromo-9,10-dioxoanthracene-2-sulphonate coupled with Aniline, subsequently coupled with p-(1,1-dimethylpropyl)phenol, subsequently coupled with Ethyl benzoylacetate, subsequently coupled with Fuming Sulfuric acid, sodium salts.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 10th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Test material information

Constituent 1

Reference substance name:

Acid Red 143

IUPAC Name:

Acid Red 143

Specific details on test material used for the study:

The test item was applied in its original form, no formulation was required.

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
- Characteristics of donor animals: approximately 7 weeks old, 1.5 – 2.5 kg. Only the eyes of healthy animals considered suitable for entry into the human food chain, were used. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Collection: head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current).
- Inspection of heads: after collection, the heads were inspected for appropriate quality.
- Stotrage: heads were wrapped with paper moistened with saline, then placed in a plastic box humidified that can be closed (4-5 heads/box).
- Transport conditions of ocular tissue: the heads were transported to testing laboratory at the earliest convenience for use. The ambient temperature was optimal (19.3 – 20.4 ºC) during the transport.
- Time interval prior to initiating testing: approximately within 2 hours from collection.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03 g

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three eyes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Eye pre-treatment: after removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors. 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.
- Examination: 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.
- Eye preparation: 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. 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 onto damp paper and the nictitating membrane was cut away with other connective tissue.
- Storage: the prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
- Apparatus: the prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position. Only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes or 0.1 to 0.15 ml/min. The door of the chamber was closed to maintain temperature and humidity.
- Examination after testing start: the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. 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 with the slit-width. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.
- Acclimatization: acclimatization started and was conducted 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.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured.

NUMBER OF REPLICATES
Each treatment group and concurrent positive control consists of three eyes. The negative control group consists of one eye.

APPLICATION DOSE AND EXPOSURE TIME
- Application apporach: stepwise approach. One out of three test item treated eyes was placed on a layer of tissue with the cornea facing upwards. After the application to the first eye, the procedure was repeated for each test item treated eye.
- Test item applkication: applied onto the centre of the cornea.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: cornea surface was rinsed thoroughly with 20 ml saline solution at ambient temperature. The gentle rinsing with 20 ml saline was performed in all imidazole and test item treated eyes after the 30, 75, 120 and 180 minutes of observation.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

NEGATIVE CONTROL
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µl.

POSITIVE CONTROL
The three positive control eyes were treated in a similar way of test item, with 0.03 g Imidazole.

METHODS FOR MEASURED ENDPOINTS
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects.
- Corneal swelling: determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope (slit-width setting: 9½, equal to 0.095 mm).
- Corneal opacity: evaluated by using the area of the cornea that is most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: fluorescein retention was measured on two occasions, baseline (t=0) and 30 minutes after the post-treatment rinse.
- Macroscopic morphological damage to the surface: no pitting of corneal epithelial cells, loosening of epithelium and roughening of the corneal surface were noted.

SCORING SYSTEM:
CORNEAL SWELLING: 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 is then given.
Mean cornea swelling and ICE Class
0 to 5, class I
> 5 to 12, class II
> 12 to 18 (> 75 min. after treatment), class II
> 12 to 18 (≤ 75 min. after treatment), class III
>18 to 26, class III
> 26 to 32 (> 75 min. after treatment), class III
> 26 to 32 (≤ 75 min. after treatment), class IV
> 32, class IV

ICE Class Categories:
No swelling I
Slight swelling II
Moderate swelling III
Severe swelling IV

CORNEAL OPACITY
Mean cornea opacity and ICE Class
0 no opacity
0.5 very faint opacity
1 scattered or diffuse areas; details of the iris are 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 maximum opacity score:
0.0 - 0.5 I
0.6 - 1.5 II
1.6 - 2.5 III
2.6 - 4.0 IV

ICE Class Categories:
No opacity I
Slight opacity II
Moderate opacity III
Severe or total opacity IV

FLUOROSCEIN RETENTION: mean fluorescein retention value of all test eyes was calculated for the 30-minute observation time point, and used for the overall category score.
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

Mean maximum opacity score:
0.0 - 0.5 I
0.6 - 1.5 II
1.6 - 2.5 III
2.6 - 3.0 IV

ICE Class Categories:
No fluorescein retention I
Slight fluorescein retention II
Moderate fluorescein retention III
Severe fluorescein retention IV

DECISION CRITERIA
Once each endpoint has been evaluated and the ICE classes has been assigned, the in vitro classification for a test chemical is assessed by reading the GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity, and fluorescein retention.

Overall in vitro classifications
No Category 3xI, 2xI, 1xII
No prediction can be made: other combinations.
Category 1 (Causes serious eye damage) 3×IV, 2×IV, 1×III, 2×IV, 1×II, 2×IV, 1×I, Corneal opacity ≥ 3 at 30 min (in at least 2 eyes), Corneal opacity = 4 at any time point (in at least 2 eyes), Severe loosening of the epithelium (in at least 1 eye).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 75 and 240 min

Value:

6 - 10

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Value:

1.7

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein retention score

Value:

1.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Using the Isolated Chicken Eye model with test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xII, 2xIII.
The substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, the test item has been categorized as “No prediction can be made”.

NEGATIVE CONTROL
The negative control NaCl (9 g/l saline) had no significant effects on the chicken eye.

POSITIVE CONTROL
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Any other information on results incl. tables

TEST ITEM

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	6 %	II
Mean maximum corneal swelling at up to 240 min	10 %	II
Mean maximum corneal opacity	1.7	III
Mean fluorescein retention	1.7	III
Other Observations	None
Overall ICE Class	1xII, 2xIII

NEGATIVE CONTROL - NaCl

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2 %	I
Mean maximum corneal swelling at up to 240 min	2 %	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	0	I
Other Observations	None
Overall ICE Class	3xI

IMIDAZOLE - Positive control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	36 %	IV
Mean maximum corneal swelling at up to 240 min	43 %	IV
Mean maximum corneal opacity	4	IV
Mean fluorescein retention	3	IV
Other Observations	Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

Applicant's summary and conclusion

Interpretation of results:

other: the result of the study is used for CLP classification within a weight of evidence approach.

Conclusions:

In vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, “No prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. 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 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.

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 and this procedure was repeated for each eye.

Adherence of the test item and the positive control Imidazole was observed on the cornea surfaces at 240 min after the post-treatment rinse.

In this ICET, test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xII, 2xIII.

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

Conclusion

Overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, “No prediction can be made”.