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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2015 to 14 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Due to the interfering colour of the test material (dark blue) the plate was also measured at 620 nm in order to more precisely evaluate the possible amount of test material remaining on the surface on the skin units and the resulting non-specific colour. This fact was considered not to adversely affect the results or integrity of the study.

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

Species:

other: EPISKIN-SM model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Model: EPISKIN-SM. The EPISKIN-SM is 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 a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-032, Expiry Date: 17 August 2015
- 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 = not acceptable). The colour of the temperature indicator was inspected to verify that the kit had 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 = good; grey or black = not acceptable). The kits were found to be in good order at reception.
- Storage: The EPISKIN-SM kit was kept in the packaging at 37 °C; the Assay Medium and Maintenance Medium supplied with the kits were stored at 2 to 8 °C until the initiation of the test.

KIT CONTENTS
- Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm²) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plate
- Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
- Medium: A flask of sterile “Maintenance Medium”, Batch No.: 15 MAIN3 032; Exp. Date: 19 August 2015. A flask of sterile “Assay Medium”, Batch No.: 15 ESSC 032; Exp. Date: 19 August 2015. A flask of sterile “Assay Medium”, Batch No.: 15 ESSC 031; Exp. Date: 12 August 2015.

KILLED EPIDERMIS
For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-022, Expiry Date: 08 June 2015) 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 06 June 2015. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

Test system

Type of coverage:

open

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Tissues were exposed to both positive (5 % (w/v) aqueous sodium dodecyl sulphate in distilled water) and negative (phosphate buffered saline) concurrent controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (test material was applied in its original form, though ground to a fine powder before use)

Duration of treatment / exposure:

15 minutes

Observation period:

After exposure the skin tissue was thoroughly rinsed to remove the test material, followed by incubation for 42 hours.

Number of animals:

Three replicates were used for the test material. Five negative controls and three positive controls were also run in the assay. As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. Furthermore, as the test material had an MTT interacting potential, three additional test material-treated killed epidermis and three negative control treated killed epidermis were used in the study.

Details on study design:

PERFORMANCE OF THE STUDY
- Pre-incubation (Day -1)
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate were 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 Material: 10 μL of distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis, and then 10 mg of test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a curved flat spatula.
Positive and negative control: 50 μL of positive control (5 % (w/v) SDS solution) or negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to evenly cover the entire epidermal surface.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (24.7 to 25.9 °C).
The test material was checked for MTT interacting potential and was found to be an MTT-interacting substance; therefore in addition to the normal procedure, three test material treated killed epidermis and three negative control treated killed epidermis were used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The same treatment steps were followed in case of the killed tissues as in case of the living tissues.
After the incubation time, the EPISKIN-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 (± 1 h) at 37 °C in an incubator with 5 % CO₂.

- MTT test (Day 2)
After the 42 hour incubation, all EPISKIN-SM units (except one colour control unit) were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 minutes) at 37 °C in an incubator with 5 % CO₂ protected from light.

- Formazan extraction (Day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (0.04 N HCl); one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

- Cell viability measurements (Day 2)
Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate. The OD (optical density or absorbance) of the samples were measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL /well) was used as blank.
In order to get more information about the amount of test material that remained on the surface of the skin units, the plate was measured additionally at 620 nm (the test material has a dark blue colour, so these wavelengths were considered to be suitable for this purpose).
The proper status of the instrument was verified by measuring a Verification plate (Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14) at the required wavelength on each day before use. Furthermore, the absorption spectrum of the test material was taken by a U-2900 spectrophotometer.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative viability percentage

Value:

85.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: n/a. Remarks: The OD values for the test material treated skin samples showed 85.9 % relative viability.. (migrated information)

In vivo

Irritant / corrosive response data:

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 1. The OD values for the test material treated skin samples showed 85.9 % relative viability.

VALIDITY OF THE TEST
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 (0.794). Standard deviation for negative control samples was 7.0.
The positive control treated tissues showed 7.2 % viability demonstrating the proper performance of the assay. The standard deviation value for positive control samples was 1.4.
The standard deviation for viability values of the three test material-treated tissue samples in the MTT assay was 9.8.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Other effects:

ADDITIONAL CONTROLS
As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. The optical density (measured at 570 nm) of this tissue was 0.105, Non Specific Colour % was calculated as 13.2%. This value was above 5 %, therefore additional data calculation was necessary.
As colour change (blue colour) was observed after three hours of incubation of the test material in MTT working solution, the test material might interact with MTT. Therefore, additional controls and data calculations were used to exclude the false estimation of viability. Based on these observed OD (0.266), the calculated NSMTT (non-specific MTT reduction) was 33.7 %.
However, the observed colour was the test material itself and it did not derive from non-specific MTT reduction based on the absorption spectra of the test material. Therefore, this value was not used for correction

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Treatment	Optical Density (OD)			TODTT	Viability (%)
	Replicate	Measured	Blank Corrected
Negative Control (PBS)	1	0.860	0.814		103.0
	2	0.804	0.758		95.9
	3	0.778	0.731		92.6
	4	0.919	0.873		110.5
	5	0.820	0.773		97.9
	Mean		0.794		100
Positive Control (5 % w/v SDS)	1	0.095	0.049		6.1
	2	0.116	0.070		8.8
	3	0.099	0.052		6.6
	Mean		0.057		7.1
Test Material	1	0.743	0.697	0.592	75.0
	2	0.853	0.807	0.702	88.9
	3	0.892	0.845	0.741	93.8
	Mean		0.783	0.678	85.9

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample.

3. TODTT: An appropriate correction for non-specific colour (subtracting the NSC value of 0.105 was applied for test material treated samples.

4. Based on the absorbance values measured at 620 nm, no significant amount of test material remained on the test material treated skin units (values in the 0.372 to 0.457 range compared to the 0.360 to 0.432 values of the negative control samples).

5. Negative control replicates #4 and #5 were washed three times.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material was determined to be non irritating to the skin.

Executive summary:

A study was performed in vitro to assess the irritancy potential of the test material in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

The in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN-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. Disks of EPISKIN (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material 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. 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. 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 (five replicates) and positive controls (three replicates), respectively.

An additional disk was used to provide an estimate of colour contribution from the test material. Furthermore, three additional test material treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test material. 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 material is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 85.9 % compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin under the conditions of this study. The experiment met the validity criteria, therefore the study was considered to be valid.