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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2017 to 14 August 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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Thiamine hydrochloride
EC Number:
200-641-8
EC Name:
Thiamine hydrochloride
Cas Number:
67-03-8
Molecular formula:
C12H17N4OS.ClH.Cl
IUPAC Name:
thiamine hydrochloride
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
- Test item storage: At room temperature protected from light and humidity

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™ (EPISKIN-SM™, 0.38 cm²)
- Tissue batch number(s): Lot no.: 17-EKIN-032
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: SkinEthic Laboratories, Lyon, France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

NUMBER OF REPLICATE TISSUES: 3

CELL CULTURE
- Tissues: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37 °C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories.
- MTT medium: MTT concentrate (3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
- Environmental conditions: All incubations, with the exception of the test material incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 % (actual range 74 - 90%), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.6 - 37.5 °C).

TEST FOR COLOUR INTERFERENCE BY THE TEST MATERIAL
The test material was checked for possible color interference before the study was started. Some non-colored test materials may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, at least 10 mg of the test material was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 10 mg of the test material was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37 °C. A negative control, 25 μL sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The test was performed on a total of 3 tissues per test material together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water to ensure close contact of the test material to the tissue and the solid test material (17.0 to 23.7 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (phosphate buffered saline, negative control) and 3 tissues with 25 μL 5 % SDS (sodium dodecyl sulphate, positive control), respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for 69.5 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate.

CALCULATION OF CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed. The corrected OD (ODc) for each sample or control were calculated by subtracting the value of blank mean (ODbl) from each reading (ODraw).
ODc = ODraw - ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc / mean ODlt_u+MTT) x 100

INTERPRETATION
- Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50 % relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

- Data evaluation and statistical procedures
A test material is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50 % of the mean viability of the negative controls.
A test material is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50 % of the mean viability of the negative controls.

≤ 50 % of the mean viability of the negative controls = Category 1 or Category 2 (additional information on corrosion needed)
> 50% of the mean viability of the negative controls = No category
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 17.0 to 23.7 mg was added into 12-well plates on top of the skin tissues

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL sodium dodecyl sulphate (SDS)
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours and then incubated for 3 hours with MTT
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
INTERFERENCE WITH MTT
- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because no colour changes were observed it was concluded that the test material did not interact with the MTT endpoint.

TISSUE VIABILITY
- Results can be seen in Tables 1 and 2.
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 94 %. Since the mean relative tissue viability for the test material was above 50 % it is considered to be non-irritant.
- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4.8 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <3 %, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin irritation test

 

A (OD570)

B (OD570)

C (OD570)

Mean (OD570) ± SD

Negative control

1.222

1.227

1.252

1.234 ± 0.016

Test material

1.172

1.170

1.135

1.159 ± 0.021

Positive control

0.032

0.087

0.059

0.059 ± 0.028

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability in the in vitro skin irritation test

 

Mean tissue viability (percentage of control)

Standard deviation (percentage)

Negative control

100

1.3

Test material

94

1.7

Positive control

4.8

2.3

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.
Executive summary:

An in vitro skin irritation test using a human skin model was carried out in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

This study tests the ability of the test material to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM™)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test material was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 94 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.

The positive control had a mean cell viability of 4.8 % after 15 ± 0.5 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 3 %, indicating that the test system functioned properly.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.