2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2017 to 16 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

EC Number:

285-413-6

EC Name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Cas Number:

85098-62-0

Molecular formula:

C30H15ClN7Na5O15S4

IUPAC Name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Test material form:

solid: particulate/powder

Remarks:

Dark brown powder

Details on test material:

- Storage conditions: Controlled room temperature (15 - 25 °C, below 70 RH%) in an opaque plastic flask.

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500 (Experiment I) and ROSS 308 (Experiment II)

Details on test animals or tissues and environmental conditions:

CHICKEN HEADS COLLECTION AND TRANSPORT
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to the Test Facility 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). The heads were received at the Test Facility and processed within 2 hours of collection in each experiment.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Number of animals or in vitro replicates:

One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

Details on study design:

SELECTION OF EYES
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.

PREPARATION OF 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.

BASELINE RECORDINGS
At the end of the acclimatisation 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 material related effect after treatment. All eyes were considered to be suitable for the assay.

EXAMINATION OF EYES AND ACCLIMATISATION 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). 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.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, 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, acclimatisation 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 acclimatisation and treatment periods.


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

NEGATIVE CONTROL USED
30 μL of physiological saline

POSITIVE CONTROL USED
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 material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material, taking care not to damage or touch the cornea.

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 BP 900® slit-lamp microscope was used for the measurements.
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

REMOVAL OF TEST SUBSTANCE
The time of application was noted, then after an exposure period of 10 seconds from the end of the application 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 after treatment and at each time point when the test item or positive control material remaining on the cornea was observed in each experiment. The test material treated eyes were rinsed additional gentle rinsing with 3x20 mL saline after treatment in each experiment.

EVALUATION
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).

Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = [FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 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 t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

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 t time 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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Run / experiment:

Experiment I

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

Experiment I

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Run / experiment:

Experiment II

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

Experiment II

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

The test material showed no significant corneal effect in the first experiment. As the test material 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (the negative control physiological saline was classified as non-irritating)
- Acceptance criteria met for positive control: yes (the positive control (imidazole) was classified as severely irritating)
The results from all eyes used met the quality control standards.
- Range of historical values if different from the ones specified in the test guideline: The negative control and positive control results were within the historical control data range in each experiment. This experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of the study, the test material was determined to be a non-irritant.

Executive summary:

An in vitro eye irritation study of the test material was performed in isolated chicken's eyes. The irritation effects of the test material were investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 438.

In each experiment 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 each experiment, three test material

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 each experiment. Thus, the experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity

change (severity 0.5) was observed on one eye. No significant fluorescein retention change (severity 0.5) was noted on three eyes. The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse. No other corneal effect was observed.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No cornea opacity change and no fluorescein retention change were noted on three eyes. The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse. No other corneal effect was observed.

Based on these in vitro eye irritation tests in isolated chicken eyes, the test material was determined to be a non-irritant.