Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2015 to 25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

EC Number:

274-999-9

EC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Cas Number:

70900-27-5

Molecular formula:

C34H32N2O8S2.2Na

IUPAC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: Blue powder
- Storage conditions of test material: Controlled room temperature (15-25 °C, below 70 RH %), protected from light and humidity

Test animals / tissue source

Species:

other: isolated chicken eyes

Strain:

other: ROSS 308 and COBB 500

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. 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 to 5 heads per box) and transported to the test site at ambient temperature. The heads were transported at the earliest convenience and processed within approximately 2 hours after receipt.
The ROSS 308 strain was used in Run I and the COBB 500 strain was used in Run II.

SELECTION AND PREPARATION OF EYES FOR THE TEST
-Eye selection
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.

- Eye examination 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). Too much pressure on the eye from the clamp was avoided. Due to 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 to 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 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, 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.

THE BASELINE ASSESSMENTS
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. Slight changes in thickness (0.0 to -1.6 %) were observed in the eyes; this is considered normal when maintaining enucleated eyes. 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.

Test system

Vehicle:

physiological saline

Controls:

other: Corneas were exposed to both positive (30 mg of powdered Imidazole) and negative (physiological saline) concurrent controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg; the test material was applied as supplied (after being further powdered).

Duration of treatment / exposure:

10 seconds from the end of the application to the cornea surface

Observation period (in vivo):

4 hours

Number of animals or in vitro replicates:

3 corneas were exposed to the test material.

Details on study design:

TEST PROCEDURE
-Treatment
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test material was applied onto the centre of the cornea, taking care not to damage or touch the cornea. The positive control eyes were treated in a similar way with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline. In Run 1 and Run 2 one eye was treated with physiological saline, three eyes with test material and another three eyes with powdered Imidazole

- Test material removal
Washing (if done): After 10 seconds the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed.
Time after start of exposure: 10 seconds from the end of the application the cornea surface

OBSERVATIONS
-Observation and assessment of corneal effects
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. A Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Results and discussion

In vivo

Results

Irritation parameter:

other: ICE Class

Basis:

mean

Remarks on result:

other: The ICE class was I

Irritant / corrosive response data:

In the first run, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by a second experiment (Run 2). The second run confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test material is not an irritant.
The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Any other information on results incl. tables

Table 1: Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change in Run 1

	Test Material		Positive Control		Negative Control
Observation	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min (%)	0.0	I	13.6	III	0.0	I
Mean maximum corneal swelling at up to 240 min (%)	0.0	I	34.3	IV	0.0	I
Mean maximum corneal opacity	0.5	I	4.00	IV	0.00	I
Mean fluorescein retention	0.33	I	3.00	IV	0.00	I
Other Observations	The test material 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.		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.		None
Overall ICE Class	3 x I		3 x IV		3 x I

Table 2: Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change in Run 2

	Test Material		Positive Control		Negative Control
Observation	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min (%)	0.6	I	14.0	III	1.6	I
Mean maximum corneal swelling at up to 240 min (%)	2.2	I	32.9	IV	1.6	I
Mean maximum corneal opacity	0.17	I	4.00	IV	0.00	I
Mean fluorescein retention	0.0	I	3.00	IV	0.00	I
Other Observations	The test material 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.		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.		None
Overall ICE Class	3 x I		3 x IV		3 x I

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material was found to be non irritating to the eye.

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

The in vitro eye irritation study was performed in isolated chicken’s eyes. Three eyes were treated with 30 mg of the test material (further powdered). The three positive control eyes were treated in a similar way with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % NaCl solution).

After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

In the first run, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by a second experiment (Run 2). The second run confirmed the negative results.

The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Under the conditions of this study the test material was found to be non irritating to the eye.