Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2017 - 04 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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitrosodiphenylamine
EC Number:
201-663-0
EC Name:
Nitrosodiphenylamine
Cas Number:
86-30-6
Molecular formula:
C12H10N2O
IUPAC Name:
N-nitroso-N-phenylaniline
Test material form:
solid: pellets

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkin Laboratories, Lyon, France
Details on animal used as source of test system:
see below
Justification for test system used:
The EpiskinTM model is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.
Based on the peer review of the results of an inter-laboratory study with the EpiskinTM model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the EpiskinTM model can be used for distinguishing between skin irritant and non-irritant chemicals. The EpiskinTM model is used for hazard identification and the classification of irritation potential within the context of the sequential skin irritation testing strategy (OECD Test Guideline No. 404, 24th April 2002 and Commission Regulation (EC), No. 440/2008, Part B.4, 30 May 2008).
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Concentration: neat (as supplied)
- Amount applied (volume or weight with unit): As the test item was a solid, 5 µL of water for injections was first applied to the surface of each epidermis to improve further contact between the test item and the tissue. Then, 10 mg (± 2 mg) of the test item were applied evenly to the surface of each tissue, taking care to spread it over the whole tissue area without damaging the tissue sample.
Duration of treatment / exposure:
Exposure period of 15 minutes, followed by rinsing.
Duration of post-treatment incubation (if applicable):
42-hour recovery period.
Number of replicates:
Three tissues were used for each item.
At the end of the viability assay, formazan level in tissues were assessed in duplicate for each tissue.
The mean OD values (mean OD) were then calculated from the six replicates on each plate.

Test system

Details on study design:
PRELIMINARY TESTS
* Test for direct MTT reduction with the test item
As a test substance may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test.
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 11 mg (± 1 mg) of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 10 µL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 5 minutes).
Then the colour of the solutions obtained was evaluated.

* Test for the detection of the colouring potential of the test item
The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 11 mg (± 1 mg) of test item to 90 µL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the colouration was evaluated.

MAIN TEST
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

PRE-INCUBATION OF THE TISSUES ON THEIR DAY OF ARRIVAL (Day 0)
A volume of 2 mL of pre-warmed (at +37°C) maintenance medium was added to 3 wells on a 12 well plate (one plate per item). Each Episkin TM tissue was then transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at +37°C, 5% CO2 in a humidified incubator for at least 24 hours.

TREATMENT OF TISSUES (Day 1)
A volume of 2 mL of pre-warmed maintenance medium was added into 3 wells on the 12 well plate for each item, respectively.
Each test or control item was then applied on three tissues for an exposure period of 15 minutes (± 1 minute) at room temperature. The items were applied topically to the corresponding tissues and gently spread over the epidermal surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time intervals to ensure each tissue received an equal exposure period.

For the positive control only, the SDS solution was re-spread after 7 minutes (± 1 minute) contact time with a curved spatula to improve the distribution of the SDS for the remainder of the contact period.

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS (Day 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

MTT VIABILITY ASSAY (Day 3)
Following the 42 hours incubation period, 2mL of a freshly prepared MTT solution (0.3 mg/mL) were added into 3 wells on each 12 well plate. The tissues were then transferred to the MTT filled wells and incubated for 3 hours (± 5 minutes) at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, a total biopsy of the epidermis was made by using the Episkin™ biopsy punch. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into micro tubes and incubated with 500 µL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C and protected from light until Day 6 of the experiment.


OPTICAL DENSITY MEASUREMENTS (Day 6)
At the end of the formazan extraction period, each tube was vortexed until the solution colour became homogenous. Each tube was used to fill 2 wells of a 96-well plate with 200 µL of extract per well. One 96 well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item.
For each 96-well plate, the average Optical Density value (OD) of 6 wells containing 200 µL of acidified isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
15 min
Run / experiment:
Mean
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE CRITERIA FOR NEGATIVE AND POSITIVE CONTROLS
- the mean cOD of the three negative controls should be = 0.6 and the Standard Deviation (SD) value of the % viability should be = 18%,
- the relative mean viability of the positive control should be = 40% of the relative mean viability of the negative control and the SD value of the % viability should be = 18%.


EVALUATION OF THE COLOURATION OF TISSUES AT THE END OF THE MTT INCUBATION PERIOD
The qualitative evaluation of the MTT staining was performed with the naked eye.
All test item and negative-treated tissues appeared blue which was considered indicative for viable tissues.

EVALUATION OF THE MTT RESULTS
All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
not skin irritating
Conclusions:
Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstructed human epidermis model.

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

Results

 

Preliminary tests

In the preliminary tests, the test item was found to have neither direct MTT reducing properties, nor colouring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 105% with a standard deviation of 4%. As the mean viability was > 50% after the MTT reduction,the results met the criteria for a non-irritant response.

 

Conclusion

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

According to the results of this study, the classification of the test item should be No Category (UN GHS and Regulation (EC) No. 1272/2008).