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

Endpoint summary

Description of key information

In the in vitro EPISKIN™(SM) model test with Hexamidine diisethionate (Batch number: 42964), the results indicate that the test item is non-corrosive to the skin.

In the in vitro EPISKINTM (SM) model test with Hexamidine diisethionate (Batch number: 42964), the results indicate that the test item is non-irritant to skin (No Category).

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.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

(29 July 2016)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”

Version / remarks:

Official Journal of the European Union No. L142 (31 May 2008)

GLP compliance:

yes

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)
*Note: No correction for purity of the test item was applied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult humanderived epidermal keratinocytes

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Details on test system:

Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-036, Expiry Date: 11 September 2017) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Number of Replicate Wells:
In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Kit Reception:
In each case, the pH of the agar medium used for transport was checked by checking thecolour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage:
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

Amount/concentration applied:

Application (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test item was applied evenly to the epidermal surface of each of two test item treated skin units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
Study code: 17/218-039B Final Report Page 15 of 29
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.4-24.6°C) covered with the plate lids.

Duration of treatment / exposure:

1 day

Duration of treatment / exposure:

1 day

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

optical density (OD) measured at 570 nm

Value:

ca. 70.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

VALIDITY OF THE TEST

The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.

The acceptable mean viability % range for positive controls is ≤ 20%. The difference of viability between the two tissue replicates should not exceed 30%.

The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Results:

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions. The mean OD value of the two negative control tissues was in the recommended range (0.654). The two positive control treated tissues showed 0.6% viability demonstrating the proper performance of the assay.

Note: One individual optical density data of the positive control (0.003) and the mean optical density data of the positive control (0.004) in this study was slightly lower than the lower limit of the historical control range (0.005). This fact had no impact on the results or integrity of the study since the positive control material showed severe effect. The difference of viability between the two test item-treated tissue samples in the MTT assay was 1.9%.

The difference of viability between the two negative control tissue samples in the MTT assay was 0.4%. The mean OD value of the blank samples (acidified isopropanol) was 0.047. All these parameters were within acceptable limits and therefore the study was considered to be valid.

INTERPRETATION OF TEST RESULTS

The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016). The cut-off value of 35% and classification method was validated in an international

validation study of this kit (Fentem, 1998).

For 2 disks:

If both disks have mean viability of ≥35% = Non Corrosive

If both disks have mean viability of <35% = Corrosive (at the corresponding incubation

period)

For more than 2 disks:

If the mean value is ≥35% and the variability is less than 50% = Non Corrosive

If the mean value is <35% and the variability is less than 50% = Corrosive

Otherwise:

If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult andthat the Study Director considers that a result is not representative.

VIABILITY RESULTS

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2. The mean OD value for the test item treated skin samples showed an 70.6% relative viability compared to the negative control (see attached illustration).

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Hexamidine diisethionate, the mean cell viability was 70.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with Hexamidine diisethionate (Batch number: 42964), the results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of Hexamidine diisethionate test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section).

The corrosivity of the test item was evaluated according to the OECD No. 431 guideline. Disks of EPISKINTM(SM) (two units) were treated with Hexamidine diisethionate test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Hexamidine diisethionate, the mean cell viability was 70.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(28 July 2015)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III,
amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

GLP compliance:

yes

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)
*No correction for purity of the test item was applied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult humanderived epidermal keratinocytes

Justification for test system used:

The EPISKINTM(SM) model has been validated for irritation testing in an international validation study [11] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Details on test system:

Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-040, Expiry Date: 09 October 2017) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994) [7]. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-033, Expiry Date: 21 August 2017) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 18 August 2017 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium).
Further use of killed tissues was similar to living tissues.

Number of Replicate Wells:
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. Furthermore, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks per test items were used.

Kit Reception:
The pH of the agar medium used for transport was checked by checking the colour of
the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent ofthe length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM (SM) kit was kept in their packaging at 37°C; the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

Amount/concentration applied:

Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Application and rinsing (Day 0)
Test Item
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

Negative and positive controls
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Note: The negative and positive controls were also part of a concurrent study (CiToxLAB study code: 17/255-043B and 17/256-043B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (25.9-26.6°C).
As the test item was shown being an MTT-interacting substance, in addition to the normal procedure, two test item treated killed epidermis and two negative control treated killed epidermis was used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.
After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis). After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Duration of treatment / exposure:

2 days

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

the optical density (OD) measured at 570 nm

Value:

ca. 90.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

VALIDITY OF THE TEST

The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.

The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.

The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.

The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Results:

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions. The mean OD value of the three negative control tissues was in the recommended

range (0.934). Standard deviation of the viability results for negative control samples was 5.2%. The positive control treated tissues showed 4.8% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.7%. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.3%. The mean OD value of the blank samples (acidified isopropanol) was 0.047. All these parameters met the acceptability criteria, therefore the study was considered to be valid.

INTERPRETATION OF TEST RESULTS

The irritation potential of test items can be classified according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals [10], and a similar system is used in CLP [12, 13]. In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item.

The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls. In case the test item is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test item is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls. The prediction model (PM) is described in the following table.

VIABILITY RESULTS

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the attached illustration. The OD values for the test item treated skin samples showed 90.3% relative viability.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Hexamidine diisethionate, the mean cell viability was 90.3% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. Based on the results of the corrosivity test (performed by CiToxLAB Hungary Ltd., study code: 17/218-039B) the test item is non-corrosive.
In conclusion, in this in vitro EPISKINTM (SM) model test with Hexamidine diisethionate (Batch number: 42964), the results indicate that the test item is non-irritant to skin (No Category).

Executive summary:

An in vitro skin irritation test of Hexamidine diisethionate test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline [1].

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated killed epidermis units and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with Hexamidine diisethionate, the mean cell viability was 90.3% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. Based on the results of the corrosivity test (performed by CiToxLAB Hungary Ltd., study code: 17/218-039B) the test item is non-corrosive.

In conclusion, in this in vitro EPISKINTM (SM) model test with Hexamidine diisethionate (Batch number: 42964), the results indicate that the test item is non-irritant to skin (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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)

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.

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

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

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

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

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

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

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

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)