2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 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

Test guideline 1

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

EC Number:

211-533-5

EC Name:

2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

Cas Number:

659-40-5

Molecular formula:

C20H26N4O2.2C2H6O4S

IUPAC Name:

4-[6-(4-carbamimidoylphenoxy)hexoxy]benzenecarboximidamide;2-hydroxyethanesulfonic acid

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White to slightly yellow powder

Specific details on test material used for the study:

Batch/Lot Number: 42964
Expiry date: 31 January 2019
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%),
protected from humidity (tight closed container)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

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, taking care not to damage or touch the cornea.

Duration of treatment / exposure:

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.

Duration of post- treatment incubation (in vitro):

Experiment I.: All cornea surfaces were cleared at 120 minutes after the posttreatment rinse.
Experiment II.:Two cornea surfaces were cleared at 30 minutes after the post-treatment rinse and one cornea surface was cleared at 75 minutes after the post-treatment rinse.

Details on study design:

THE BASELINE ASSESSMENTS
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 -45min 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.

TEST PROCEDURE
Treatment
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.
In each experiment negative control eye was treated with 30 μL of physiological saline;positive control eyes were treated with 30 mg powdered imidazole.One eye was treated with physiological saline, three eyes with the test item and another three eyes with powdered imidazole in each experiment.

Test item removal
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 item treated eyes were rinsed additional gentle rinsing with at least 4x20 mL saline after treatment in each experiment.
Note: Physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 72034Y05-1, Expiry date: 30 April 2020) was used for rinsing in each experiment.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling at up to 75 min

Run / experiment:

I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling at up to 240 min

Run / experiment:

I

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

I

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

I

Value:

0.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE = II

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling at up to 75 min

Run / experiment:

II

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling at up to 240 min

Run / experiment:

II

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

II

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

II

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE = II

Other effects / acceptance of results:

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.

Any other information on results incl. tables

The test item Hexamidine diisethionate 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 with Hexamidine diisethionate, the test item was non-irritant, UN GHS Classification: No Category.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these in vitro eye irritation in the isolated chicken eyes tests with Hexamidine diisethionate, the test item was non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

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 the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined in each experiment.

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 corneal opacity

change (severity 0.5 or 0.0) was noted on three eyes. Slight fluorescein retention change (severity 1 or 0.5) was noted on three eyes. The test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 120 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 significant cornea opacity change (severity 0.5) was noted on three eyes. Slight fluorescein retention change was observed (severity 1) on three eyes. The test item was stuck on all cornea surfaces after the post-treatment rinse. Two cornea surfaces were cleared at 30 minutes after the post-treatment rinse and one cornea surface was cleared at 75 minutes after the post-treatment rinse. No other corneal effect was observed.