Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2016 to 29 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 guidelineopen allclose all
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Reaction mass of trisodium4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-4-yl]azo]benzenesulphonate and trisodium4-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-4-yl]azo]benzenesulphonate
Molecular formula:
C25H15Cl3N9Na3O10S3 (both components have the same molecular formula)
IUPAC Name:
Reaction mass of trisodium4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-4-yl]azo]benzenesulphonate and trisodium4-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-4-yl]azo]benzenesulphonate
Test material form:
solid: particulate/powder
Details on test material:
- Physical Appearance: Orange powder
- Storage conditions: Controlled room temperature (15 - 25 °C, below 70 RH %) and protected from light

Test animals / tissue source

Species:
other: Isolated chicken eyes
Strain:
other: COBB 500 and ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and heads transported to the testing laboratory at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: The heads were received at the test site and processed within 2 hours and 15 minutes of collection.
- 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 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein treated cornea was examined with a 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item.
Duration of treatment / exposure:
10 seconds from the end of the application to the cornea surface.
Observation period (in vivo):
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Duration of post- treatment incubation (in vitro):
240 minutes (4 hours)
Number of animals or in vitro replicates:
3 corneas were exposed to the test material.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, 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.
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 avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

EQUILIBRATION AND BASELINE RECORDINGS
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological 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, 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. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatization and treatment periods.
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 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

NEGATIVE CONTROL USED
In each experiment negative control eye was treated with 30 μL of physiological saline.

POSITIVE CONTROL USED
Positive control eyes were treated with 30 mg powdered Imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
An exposure period of 10 seconds from the end of the application was used.

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 2x20 mL saline after treatment in each experiment.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated according to the following formulae:
ΔCO at time t = CO at time t – CO at t = 0
Mean ΔCOmax = [FECOmax(30 to 240 min) + SECOmax(30 to 240 min) + TECOmax(30 to 240 min)] / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t = 0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at time t and corneal opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO = third eye cornea opacity
max(30 to 240 min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline corneal opacity of the individual eye

- Damage to epithelium based on fluorescein retention:
Fluorescein retention was calculated according to the following formulae:
ΔFR at time t = FR at time t – FR at t = 0
Mean ΔFR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t = 0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at time t and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

- Swelling:
Corneal swelling was calculated according to the following formulae:
CS at time t = [(CT at time t –CT at t = 0) / CT at t = 0] x100
Mean CS at time t = [FECS(at time t) + SECS(at time t) + TECS(at time t)] / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) 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 (class II).

Results and discussion

In vitro

Results
Irritation parameter:
other: ICE Class
Run / experiment:
Mean
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Acceptance criteria met for negative control: The positive control (Imidazole) was determined to be severely irritating. Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse. Overall ICE Class: 1 x III, 2 x IV in both experiments.

- Acceptance criteria met for positive control: The negative control physiological saline was determined to be non-irritating. Overall ICE Class: 3 x I in both experiments.

- Range of historical values if different from the ones specified in the test guideline: The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

In vivo

Irritant / corrosive response data:
The test material showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test material was non-irritant.
In both experiments test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse. Overall ICE Class: 2 x I, 1 x II in both experiments.

Any other information on results incl. tables

Validity

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the test, the test material was determined to be a non-irritant.
Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions.

After the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid.

For Experiment 1, no significant corneal swelling (mean ≤5 %) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

For Experiment 2, no significant corneal swelling (mean ≤5 %) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Under the conditions of the test, the test item was determined to be a non-irritant based on the in vitro eye irritation assays in isolated chicken eyes.