Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August-10 October, 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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Expiration date:26 March 2020

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
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
no- 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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 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.
- Tissue batch number: 16-EKIN-037

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the epidermis model was 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.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: water killed
- N. of replicates: 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color 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.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50 % of the negative control.





Control samples:
yes, concurrent negative control
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg


NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 %
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 h (± 1 h)
Number of replicates:
In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color 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
Remarks:
mean
Run / experiment:
mean (Replicate 1-3)
Value:
100
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 1
Value:
116
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 2
Value:
97
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 3
Value:
89
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
No
- Direct-MTT reduction:
No
- Colour interference with MTT:
Yes

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes
- Acceptance criteria met for positive control:
Yes
- Acceptance criteria met for variability between replicate measurements:
Yes

Any other information on results incl. tables

Table 2: OD values and viability percentages of the controls

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.018

117

2

0.823

95

3

0.765

88

mean

0.869

100

standard deviation (SD)

15.22

Positive Control:
SDS (5 % aq.)

1

0.167

19

2

0.168

19

3

0.072

8

mean

0.136

16

standard deviation (SD)

6.34

Table 3: OD values and viability percentages of the test item

Test Item

Optical Density (OD)

Viability (%)

C. I. Leuco Sulfur Green 2

1

1.004

116

2

0.845

97

3

0.769

89

mean

0.873

100

standard deviation (SD)

13.84

 

Table 4: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.065

2

0.078

3

0.079

mean

0.074

Test item treated killed tissues:
C. I. Leuco Sulfur Green 2

1

0.042

2

0.038

3

0.030

mean

0.037

 

Table 5: OD values and NSC % of additional control

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

C. I. Leuco Sulfur Green 2
(test item treated tissueswithout MTT incubation)

1

0.020

2.3

2

0.020

mean

0.020


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation assay according to OECD guideline 439, the test item did not show a skin irritation potential.

Executive summary:

An in vitro skin irritation assay according to OECD guideline 439 was performed to determine the skin irritating potential of the test item. Disks of EPISKIN (three units) were treated with 10 mg of the 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% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1x PBS treated (three units / positive and negative control) tissues were used as positive or negative controls respectively. For each treated tissue viability was expressed as percentage relative to negative control.

The test item has an intrinsic colour (bluish black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. Additionally, the test item is 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.

Since the test item is a possible MTT-reducer and has an intrinsic colour (bluish black) a third control for non-specific colour in killed tissues (NSCkilled) was performed, to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference. Two killed treated tissues were used to avoid a possible double correction for colour interference. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item did not show significantly reduced cell viability compared to the negative control (mean viability: 100 %). Therefore the test item was considered to be non-irritant to skin. 

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the testing conditions. The test item is considered to be non-irritant to skin and therefore no classification is warrant (UN GHS No Category).