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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2016 to 12 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Reaction mass of trisodium 4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate and trisodium 4-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate
EC Number:
943-063-8
Molecular formula:
C25H15Cl3N9Na3O10S3 (both components have the same molecular formula)
IUPAC Name:
Reaction mass of trisodium 4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate and trisodium 4-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate
Test material form:
solid: particulate/powder
Details on test material:
- Physical Appearance: Orange powder
- Storage conditions: Controlled room temperature (15 - 25 °C, below 70 RH %) and protected from light

In vitro test system

Test system:
human skin model
Remarks:
EPISKIN™ (SM) reconstituted human epidermis (three-dimensional human epidermis model)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
other: Not applicable
Details on animal used as source of test system:
SOURCE ANIMAL
Not applicable; EPISKIN™ (SM) reconstituted human epidermis (three-dimensional human epidermis model).
Justification for test system used:
The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study 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.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 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.
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). The kits were found to be in good order.
The EPISKIN™ (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.
Procedures described were performed under aseptic conditions (in sterile hood using sterile equipment).

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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (24.4 – 26.4 °C).
- Temperature of post-treatment incubation: 37 °C
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.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. If the test item was stuck on the surface of the epidermis additional rinsing was used. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: None specified
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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: After the 42 hours incubation, all EPISKIN™ (SM) units (except of the two living colour control units) were transferred into the MTT working solution filled with 2 mL of 0.3 mg/mL MTT per well.
- Incubation time: 3 hours (± 5 mins) at 37 °C with 5 % CO2 protected from light.
- Wavelength: 570 nm
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 09 August 2016) was stored in refrigerator (2-8 °C) protected from light. It was diluted with pre-warmed (37 °C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

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 (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.

Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

NUMBER OF REPLICATE TISSUES: 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 items were coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls are used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are as follows:
Approximately 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: Yellow
-Test items reacting with MTT: Blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.
Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test items had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.

PREDICTION MODEL / DECISION CRITERIA
The test item is considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg of the test item was applied evenly to the epidermal surface. The amount was sufficient to cover the epidermal surface.

NEGATIVE CONTROL
- Amount applied: 50 μL of negative control (Phosphate Buffered Saline (PBS)) was added to each skin unit by using a suitable pipette. The negative control was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

POSITIVE CONTROL
- Amount(s) applied: The positive control 5 % (w/v) Sodium Dodecyl Sulphate solution was added to each skin unit by using a suitable pipette. The positive control was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- Preparation: The positive control solution was prepared freshly in the testing laboratory.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2.
Number of replicates:
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.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: Relative viability percentage
Run / experiment:
Mean
Value:
96.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None specified
- Direct-MTT reduction: As no colour change (yellow colour) was observed after three hours of incubation of the test items in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Colour interference: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.015, Non Specific Colour % was calculated as 2.0 %. This value was below 5 %, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
The OD values for the test item treated skin samples showed 96.9 % relative viability. This is above the threshold of 50 % and therefore the test item was considered non-irritant to the skin.
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.748). Standard deviation of the viability results for negative control samples was 9.1.
- Acceptance criteria met for positive control: The positive control treated tissues showed 8.8 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 4.2.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 10.2. The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study the test material is non-irritant to 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.

Disks of EPISKIN (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. 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 and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. 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 the test material, the mean cell viability was 96.9 % 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.

Under the conditions of the study the test material is non-irritant to skin.