Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2017 - 05 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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test Item: Leuco Sulfur Blue 15
Appearance: Dark, near black 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 model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and non-skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, 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 irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Supplier: SKINETHIC Laboratories, 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 17-EKIN-020
Expiry date: 22 May 2017

Units: EpiSkinTMSM 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: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: sterile “Maintenance Medium” for incubations (Batch No.: 17 MAIN3 020; Exp. Date: 24 May 2017)
sterile “Assay Medium” for use in MTT assays (Batch No.: 17 ESSC 019; Exp. Date: 24 May 2017)

The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay media were stored at 2-8°C.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 mg of the test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of 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, ≥ 95 % humidified atmosphere.

Number of replicates:
In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
1-3
Value:
103
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:
Validity of the Test

The mean OD value of the three negative control tissues was 0.614. Furthermore, the individual OD values of the three negative control tissues were in the acceptable range (0.603, 0.637 and 0.602). The mean OD value obtained for the positive control was 0.125 and this result corresponds to 20 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for potential false viability

Possible direct MTT reduction with test item:
As the test item has an intrinsic colour (black), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.
The non-specific MTT reduction (NSMTT) was determined (0%)*, the correction of viability percentage was not necessary.
*: The calculated NSMTT was -1.322%. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

Colouring potential of test item:
As the test item has an intrinsic colour (black), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.014. The Non Specific Colour % (NSC %) was calculated as 2.3 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells are presented below:

 

Table 1: OD values and viability percentages of the controls:

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

0.603

98

2

0.637

104

3

0.602

98

mean

0.614

100

standard deviation (SD)

3.23

Positive Control:
SDS (5 % aq.)

1

0.139

23

2

0.096

16

3

0.138

23

mean

0.125

20

standard deviation (SD)

3.99

 

Table 2: OD values and viability percentages of the test item:

Test Item

Optical Density (OD)

Viability (%)

1

0.641

104

2

0.646

105

3

0.616

100

mean

0.634

103

standard deviation (SD)

2.65

 

Table 3: OD values of additional controls for MTT-interacting test item:

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.083

2

0.039

3

0.073

mean

0.065

Test item treated killed tissues:

1

0.069

2

0.051

3

0.050

mean

0.057

 

Table 4: OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

Test item
(test item treated tissueswithout MTT incubation)

1

0.020

2.3

2

0.008

mean

0.014

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

EpiSkinTMSM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. 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 5% CO2protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is also a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. As the test item is a possible MTT-reducer and has an intrinsic colour (black) to avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 103 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).