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

Skin irritation / corrosion

Administrative data

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

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

Version / remarks:

(29 July 2016)

Test guideline 2

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

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

In vitro test system

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

Test system

Duration of treatment / exposure:

1 day

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.