2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 31, 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 Ocular Corrosives and Severe Irritants)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yelow 236

IUPAC Name:

Acid Yelow 236

Specific details on test material used for the study:

The test item was finely ground before application.

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.
- 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).
- Health inspection: after collection, the heads were inspected for appropriate quality.
- Storage, temperature and transport conditions of ocular tissue: after collection, heads were wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box). The ambient temperature was optimal (19.4 – 20.3 ºC) during the transport.
- Time interval prior to initiating testing: use approximately within 2 hours from collection.
- Eyes: all eyes used in the assay were from the same groups of eyes collected on one specific day.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

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

Controls:

yes

Amount / concentration applied:

The subsatnce was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

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

Number of animals or in vitro replicates:

Each treatment group and concurrent positive control consists of 3 eyes. The negative control group consists of 1 eye.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eye selection
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.

Preparation of Eyes
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. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes Examination and Acclimatization Time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, 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 except for manipulations and examinations, to maintain temperature and humidity.
The flow of saline to the eyes by the mechanical pump was monitored.
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. 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. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NEGATIVE CONTROL USED
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µl from micropipette, in such a way that the entire surface of the cornea was covered with negative control.

POSITIVE CONTROL USED
Three positive control eyes were treated in a similar way as for test item, appling 0.03 g of imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea.

REMOVAL OF TEST SUBSTANCE
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 and test item 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.

OBSERVATIONS
The cornea thickness, cornea opacity and morphological effects (e.g., pitting or loosening of the epithelium) were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

Retention of Corneas: at the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

METHODS FOR MEASURED ENDPOINTS and SCORING SYSTEM

CORNEA SWELLING
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope (slit-width setting: 9½, equal to 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 is then given.
For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5 %) following application are counted as zero. Large negative numbers (> -12 %) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

Scoring system - ICE classification criteria for corneal swelling
Mean Corneal Swelling (%) // ICE Class
0 to 5 I
> 5 to 12 II
> 12 to 18 ( >75 min after treatment ) II
> 12 to 18 ( <75 min after treatment ) III
> 18 to 26 III
> 26 to 32 ( >75 min after treatment ) III
> 26 to 32 ( <75 min after treatment ) IV
> 32 IV

The four categories of ICE Class are:
- No swelling I
- Slight swelling II
- Moderate swelling III
- Severe swelling IV

CORNEA OPACITY
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal opacity was evaluated by using the area of the cornea that is most densely opacified for scoring.
Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score is then given.

Scoring system - ICE classification criteria for corneal opacity
Corneal opacity score:
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 // ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 4.0 IV

The four categories of ICE Class are:
- No swelling I
- Slight swelling II
- Moderate swelling III
- Severe swelling IV

FLUORESCEIN RETENTION
The fluorescein retention was measured on two occasions, baseline (t=0) and 30 minutes after the post-treatment rinse.
The mean fluorescein retention value of all test eyes was calculated for the 30-minute observation time point, and used for the overall category score.

Scoring system - ICE classification criteria for fluorescein retention
Flurescein retention 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 fluorescein retention score // ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 3.0 IV

The four categories of ICE Class are:
- No swelling I
- Slight swelling II
- Moderate swelling III
- Severe swelling IV

INTERPRETATION OF RESULTS
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 as described below.

UN GHS Classification, combinations of the 3 Endpoints
- No Category:
3 x I
2 x I,1 x II
- Category 1 (Causes serious eye damage)
3 × IV
2 × IV, 1 × III
2 × IV, 1 ×II (combinations of categories less likely to occur)
2 × IV, 1 × I (combinations of categories less likely to occur)
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)
- No prediction can be made: other combinations

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item did not cause ocular corrosion or severe irritation (overall ICE class: 1xI, 1xII, 1xIV)

Other effects / acceptance of results:

Under tested consitions, test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE score was 1xI, 1xII, 1xIV.

According to the guideline OECD 438, the overall 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, the test item has been categorized as “No prediction can be made”.

No pitting of corneal epithelial cells, loosening of epithelium, roughening of the corneal surface and sticking of the test substance to the cornea were noted.

CONTROLS
Positive and negative control values were within the corresponding historical control data ranges.
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
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: neither classified as an ocular corrosive nor severe eye irritant, nor not classified.

Conclusions:

The substance overall in vitro classification is neither classified as an ocular corrosive or severe eye irritant, nor not classified.

Executive summary:

The Isolated Chicken Eye Test (ICET) system was used 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 in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. 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.

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.

Under test conditions, the substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 1xII, 1xIV. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Conclusion

According to the guideline OECD 438, the substance overall 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, the test item has been categorized as “No prediction can be made”.