Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 July 2017 to 14 July 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 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
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile
EC Number:
250-654-8
EC Name:
3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile
Cas Number:
31482-56-1
Molecular formula:
C17H17N5O2
IUPAC Name:
3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
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™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic, France.
- Tissue lot number: 17-EKIN-028
- Expiry date: 17 July 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.8 - 27.3°C)
- Temperature of post-treatment incubation: 37°C

ASSESSMENT OF POSSIBLE MTT REDUCTION
10 mg of test material 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.
After three hours incubation, red colour was detected in the test tube. Thus, the test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOURING POTENTIAL OF TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or extracting solution. The test material 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 the test material to stain the epidermis by using additional control tissues.

Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

MAIN STUDY

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 OF TEST MATERIAL (DAY 0)
10 μL distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test material was applied evenly to the epidermal surface. The test material was spread gently on the skin surface with a pipette tip without damaging the epidermis.
50 μL of negative control or positive control were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly without damaging the epidermis.

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period, each tissue was removed from the well and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface. 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.

MTT TEST (DAY 2)
After the 42 hours incubation, all tissues (except of two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred tissues were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

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 (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.

NUMBER OF REPLICATE TISSUES: 3

CELL VIABILITY MEASUREMENTS
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.
The proper status of the instrument was verified by measuring a Verification plate at the required wavelength on each day before use.



Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to:
- Relative mean tissue viability is ≤ 50%: Irritant (H315 Category 2)
- Relative mean tissue viability is > 50%: Non-irritant (Not classified for irritation)

-Quality criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤ 40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤ 18%.
- Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
- Test Material: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (following 10 µL distilled water applied to epidermal surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours in 2 mL/well Maintenance Medium. After the 42 hours incubation, all EPISKIN™ (SM) units (except the two living colour control units were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well) and incubated for a further 3 hours.
Number of replicates:
Three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
88.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Result is above the threshold of 50% indicating the test material is non-irritant to the skin
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: As no colour change (red colour) was observed after three hours of incubation of the test material in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded. As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.035, Non Specific Colour % was calculated as 5.0%. This value was equal 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean corrected OD value of the three negative control tissues was in the recommended range (0.694). Standard deviation of the viability results for negative control samples was 2.6 (%).
- Acceptance criteria met for positive control: The positive control treated tissues showed 5.9% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.5 (%).
- Range of historical values if different from the ones specified in the test guideline: Negative control (OD) - 0.573-1.362 (mean 0.802); Positive control (OD) - 0.032-0.354 (mean 0.094)

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of the study, the in vitro EPISKIN™ (SM) test indicated that the test material is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of the test material was performed in a reconstructed human epidermis model EPISKIN™ (SM), under GLP conditions and according to the standardised guidelines OECD 439 and EU Method B.46.

During the study, disks of EPISKIN™ (SM) (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 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 in a >95% humidified atmosphere 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 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 88.3% compared to the negative control. This is above the threshold of 50%, therefore the test mateial was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.