Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-11 to 2017-01-30
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Deviation was not considered to have affected the integrity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Source strain:
not specified
Justification for test system used:
In vitro study - The purpose of the study was to assess the potential skin irritancy of the test material as measured by its ability to induce cell death in a commercial reconstructed human epidermis (RhE)model, EPISKIN™.

The test system EPISKIN™ is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.
Vehicle:
not specified
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE - MTT Method
- Model used: EPISKIN™ - 0.38 cm2
- Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
- Tissue batch number(s): 16-EKIN-049, 17-EKIN-004
- Delivery date: 06 Dec 2016 and 24 Jan 2017
- Date of initiation of testing: 11 November 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.
- Observable damage in the tissue due to washing: no damage
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL/well of MTT ready-to-use solution.
- Incubation time: 3hr
- Wavelength: spectral analysis at 595 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 15 ± 0.5 minutes exposure is less than 50%
- The test substance is considered to be non-corrosive to skin if the viability after 15 ± 0.5 minutes exposure is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
NEGATIVE CONTROL (Live Tissue)
- Amount(s) applied (volume or weight): 20 µL

POSITIVE CONTROL (Live Tissue)
- Amount(s) applied (volume or weight): 20 µL

TEST MATERIAL (Live Tissue)
- Amount(s) applied (volume or weight with unit): 20 mg

TEST MATERIAL (Without MTT Live Tissue)
- Amount(s) applied (volume or weight with unit): 20 mg

TEST MATERIAL (Killed Tissue)
- Amount(s) applied (volume or weight with unit): 20 mg

NEGATIVE CONTROL (Killed Tissue)
- Amount(s) applied (volume or weight): 20 µL

TEST MATERIAL (Without MTT (Killed Tissue)
- Amount(s) applied (volume or weight with unit): 20 mg




Duration of treatment / exposure:
An exposure time of 15 ±0.5 minutes was allowed in a ventilated cabinet at room temperature.
Duration of post-treatment incubation (if applicable):
A 42 ± 1 hour recovery period was allowed by incubation at 37 °C, 5% CO2 and saturated humidity.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Test Material
Run / experiment:
2
Value:
ca. 98
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Preliminary test
Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system.
 
In a first step, the test item was assayed for the ability of reducing MTT per se. A grey purple solution was observed at the end of the incubation period, indicating that the test item could direct interact with MTT.
 
In a second step, the test item was assayed for the ability of colouring water per se and spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. A colourless solution was
observed; however, the value obtained for the Optical Density (OD) was 0.217, indicating that the test item has a potential interfering ability. Based on the results obtained, additional controls were added in the Main Assay for the evaluation of Non Specific Colouring potential (NSCliving) and Non Specific MTT reduction (NSMTT). Since the test item was able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.

- OTHER EFFECTS:
- Direct-MTT reduction: In the preliminary test, a grey purple solution was observed at the end of the incubation period, indicating that the test item coulddirect interact with MTT.
- Colour interference with MTT: no relevant colouring ability of the test item was noted

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications).
- Acceptance criteria met for positive control: The positive control caused the expected cell death (mean viability ≤40%) and variability(SD of % viability lower than 18).
- Acceptance criteria met for variability between replicate measurements: yes (Acceptable intra-replicate variability was obtained (SD of % viability = 4.5 lower than 18)

Any other information on results incl. tables

Table 2. Preliminary Test – Results - Direct MTT reduction test (Step 1)

Test Item (mg)

MTT ready to use solution (mL)

Container

Incubation condition

Colour Observation

20.0

2.0

Well

3 h at 37°C

100% nominal humidity 5% CO2

Grey purple colour (possible interaction)

 

Table 3. Preliminary Test – Results - Colouring potential test (Step 2)

Test Item (mg)

Water (µL)

Container

Incubation condition

Observation

20

180

Eppendorf Tube

15’, ambient condition, in agitation

Colourless solution; spectrophotometer analysis: OD=0.217

(possible interaction)

Table 4. Main Assay - Results

Sample

 

Optical Density (OD)

Viability (%) (mean)

NSMTTkilled

(%)

NSCkilled

(%)

NSCliving(%)

Run 1

 

Negative control

(live tissue)

Mean

0.6

100

-

-

-

SD

0.04

6.7

-

-

-

CV%

6.7

6.7

-

-

-

 

Negative control

(killed tissues)

Mean

0.0

-

-

-

-

SD

0.00

-

-

-

-

CV%

10.4

-

-

-

-

 

Test Item

(killed tissues)

Mean

0.0

-

-1

-

-

SD

0.02

-

2.83

-

-

CV%

48.7

-

-392.8

-

-

 

Test Item

(killed tissues without MTT)

Mean

0.0

-

-

1

-

SD

0.00

-

-

0.1

-

CV%

4.7

-

-

4.7

-

Run 2

 

Negative control

Mean

0.9

100

-

-

 

SD

0.04

4.2

-

-

 

CV%

4.2

4.2

-

-

 

 

Positive control

Mean

0.0

4

 

 

 

SD

0.01

0.6

 

 

 

CV%

16.1

16.1

 

 

 

 

Test Item

Mean

0.8

98

 

 

 

SD

0.04

4.5

 

 

 

CV%

4.6

4.6

 

 

 

 

Test Item

(without MTT)

Mean

0.0

-

-

-

0

SD

0.00

-

-

-

0.1

CV%

18.4

-

-

-

18.4

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to the CLP Regulation
Remarks:
not irritating to the skin.
Conclusions:
Based on the results observed, the test material does not meet the GHS classification criteria for skin irritation.
Executive summary:

In a key Guideline (OECD 439) in vitro skin irritation study, the potential of the test material to be irritant to the skin was investigated using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The test material, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period.

A preliminary test was carried out to evaluate the compatibility of the test material with the test system. In the first step, the test material was assayed for to evaluate its ability to reduce MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS N. 298-93-1). A grey purple solution was observed at the end of the incubation period, indicating that the test material could directly interact with MTT. In the second step, the test material was assayed for the ability of colouring water and spectral analysis of the test material in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. A colourless solution was observed, however the value obtained for the Optical Density (OD) was 0.217, indicating that the test material has a potential interfering ability. Based on these results, additional controls were added in the Main Assay.

In the Main Assay, the test material was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2(treatment level: 53 mg/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl

sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20μL/epidermis unit. To verify if the test material results had to be corrected, the non-specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non-specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test material was able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.

Data presented for the alive tissues treated with the test material were obtained in a repeated experiment (second run). In the first run, probably due to a microbial contamination in two epidermis units, unacceptable variability within replicate tissues

stained with MTT was observed. Since the non-specific colour (NSCliving) control needs to be performed concurrently to the testing of the coloured test chemical, this control was also repeated. In each run, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

 

The positive control caused expected cell death (mean viability ≤40%) and variability (SD of % viability lower than 18). Based on the stated criteria, the assay was regarded as valid. Following treatment of alive tissue without MTT, no relevant colouring

ability of the test material was noted; no relevant interaction was recorded between the test material and MTT. Based on these results, only the OD-blank background subtraction was performed. In the second run, the test material did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 98%, when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 4.5 (lower than 18,as stated in the Study Protocol).

 

Based on the results observed, the test material is considered as not irritating to the skin.