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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2016 - 01 March 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)
Version / remarks:
2015 July 28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012 July 06
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide
EC Number:
600-519-8
Cas Number:
1040873-93-5
Molecular formula:
not applicable
IUPAC Name:
Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide
Test material form:
solid: particulate/powder
Details on test material:
Test item: Blue Sema
Appearance: Black to brownish black, solid
CAS No: 1040873-93-5
EC No: 600-519-8

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.
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).
Duration of post-treatment incubation (if applicable):
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, ≥ 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

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
1-3
Value:
96
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 1.048. The mean OD value obtained for the positive control was 0.116 and this result corresponds to 11 % 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 to brownish 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 to be 0.488 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

Colouring potential of test item:
As the test item has an intrinsic colour (black to brownish 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 0.010. The Non Specific Colour % (NSC %) was calculated as 0.9 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

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

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.135917

108

2

0.909867

87

3

1.096867

105

mean

1.047550

100

standard deviation (SD)

11.53

Positive Control:
SDS (5 % aq.)

1

0.053267

5

2

0.090567

9

3

0.203817

19

mean

0.115883

11

standard deviation (SD)

7.48

Table 2: OD values and viability percentages of the test item (including corrected values):

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

1

0.980617

0.975500

94

93

2

1.090367

1.085250

104

104

3

0.957067

0.951950

91

91

mean

1.009350

1.004233

96

96

standard deviation (SD)

6.79

6.79

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

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.022267

2

0.026317

3

0.030267

mean

0.026283

Test item treated killed tissues:

1

0.025767

2

0.032567

3

0.035867

mean

0.031400

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

0.009117

0.9

2

0.010217

mean

0.009667


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is determined not to be 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 3 7°C in 5% CO2 protected 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 to brownish 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 brownish black) to avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues 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: 96 %). 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).

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