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Skin irritation / corrosion

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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:
08 May 2019 - 08 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reference substance 001
Cas Number:
67-47-0
Molecular formula:
C6H6O3
impurity 1
Chemical structure
Reference substance name:
Fructose
EC Number:
200-333-3
EC Name:
Fructose
Cas Number:
57-48-7
Molecular formula:
C6H12O6
IUPAC Name:
D-fructose
impurity 2
Chemical structure
Reference substance name:
1-(furan-2-yl)-2-hydroxyethanone
Cas Number:
17678-19-2
Molecular formula:
C6H6O3
IUPAC Name:
1-(furan-2-yl)-2-hydroxyethanone
impurity 3
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
impurity 4
Chemical structure
Reference substance name:
Sodium formate
EC Number:
205-488-0
EC Name:
Sodium formate
Cas Number:
141-53-7
Molecular formula:
CH2O2.Na
IUPAC Name:
sodium formate
impurity 5
Chemical structure
Reference substance name:
Sodium nitrate
EC Number:
231-554-3
EC Name:
Sodium nitrate
Cas Number:
7631-99-4
Molecular formula:
HNO3.Na
IUPAC Name:
sodium nitrate
impurity 6
Chemical structure
Reference substance name:
Glucose
EC Number:
200-075-1
EC Name:
Glucose
Cas Number:
50-99-7
Molecular formula:
C6H12O6
IUPAC Name:
D-glucose
impurity 7
Chemical structure
Reference substance name:
Sodium acetate
EC Number:
204-823-8
EC Name:
Sodium acetate
Cas Number:
127-09-3
Molecular formula:
C2H4O2.Na
IUPAC Name:
sodium acetate
impurity 8
Chemical structure
Reference substance name:
2-furaldehyde
EC Number:
202-627-7
EC Name:
2-furaldehyde
Cas Number:
98-01-1
Molecular formula:
C5H4O2
IUPAC Name:
2-furaldehyde
impurity 9
Chemical structure
Reference substance name:
Sodium 4-oxovalerate
EC Number:
243-378-4
EC Name:
Sodium 4-oxovalerate
Cas Number:
19856-23-6
Molecular formula:
C5H8O3.Na
IUPAC Name:
sodium 4-oxopentanoate
impurity 10
Chemical structure
Reference substance name:
Formaldehyde
EC Number:
200-001-8
EC Name:
Formaldehyde
Cas Number:
50-00-0
Molecular formula:
CH2O
IUPAC Name:
formaldehyde
Test material form:
solid: crystalline
Details on test material:
freeze-dried material
Batch: 1808A-RO-C-I1-FD-Chg#3
Appearance Crystal / brown
Storage: Refrigerator (2 - 8 °C)
Expiring date: 31 December 2019
Specific details on test material used for the study:
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage Conditions: 2-8°C

In vitro test system

Test system:
human skin model
Remarks:
EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France
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. The EpiSkin method is 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) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Remarks:
epidermal surface was first moistened with 5 µL deionised water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number: 19-EKIN-019
- Expiry date: 13 May 2019
- Date of initiation of testing: 08 May 2019

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated over night at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE (DAY 0)
- Test Item: The epidermal surface was first moistened with 5 µL deionised water (prepared by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C in Toxi-Coop ZRT) in order to improve further contact between the test item and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
- Positive and negative control: A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Triplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1x solution, once. 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: none

POST-INCUBATION (DAY 0-2)
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.

MTT TEST (DAY 2)
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5±1 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION (DAY 2)
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS (DAY 2)
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA
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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg of the test item.

POSITIVE AND NEGATIVE CONTROL
- Amounts applied: A volume of 10 µL positive or negative control.
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 ± 1h
Number of replicates:
Three replicates were used for the test item and controls, respectively.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
72
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean corrected value
Other effects / acceptance of results:
OTHER EFFECTS:
- Possible direct MTT reduction with test item: As the test item has an intrinsic colour (brown), the check-method for possible direct MTT reduction with test item was not completed for the following reason: The color of the MTT solution was close to black after it was mixed with test item. So the interaction of the test item with MTT solution could not be determined. Based on this information the additional controls were automatically used. This step prevents the false viability which is the possible interaction with MTT, and it occurs if the test item cannot be removed totally from the surface of the tissues. In this situation the direct interaction with MTT was defined based on the OD results of additional controls.
The non-specific MTT reduction (NSMTT) was determined to be 5.329 %. 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: The test item has an intrinsic colour (brown). If the test item is not white, off withe, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test item. 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 0.040. The Non Specific Colourliving % (NSCliving %) was calculated as 4.8 %. As the NSCliving is below 5%, true MTT metabolic conversion for colour interference is not undertaken.

ACCPETANCE OF RESULTS
- Acceptance criteria met for negative control: Mean OD value 0.834 and standard deviation value (SD) for the % viability 0.43 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 20% mean viability range standard deviation value (SD) for the % viability 7.27 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 6.57 % SD

Any other information on results incl. tables

OD values and viability percentages of the controls:

 Control  Optical Density (OD)     Viability (%)

 Negative Control            

1X PBS

 1  0.0831  100
 2  0.0833   100
 3  0.0838   100
 mean  0.0834   100
 standard deviation (SD)     0.43

Positive Control

SDS (5% aq.)             

 1  0.136  16
 2  0.136  16
 3  0.241  29
 mean  0.171  20
    standard deviation (SD)  7.27

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

 Test Item  Optical Density (OD)     TODTT  Viability (%)  realtive Viability (%)
5-(Hydroxymethyl)
furfural
            
 1  0.668  0.623  80  75
 2  0.581  0.536  70  64
 3  0.682  0.637  82  76
 mean  0.643  0.599  77  72
 standard deviation (SD)        6.57  6.57

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

 Additional controls  Optical Density (OD)   
 

Negative control killed tissues:

1x PBS

1

2

3

mean

0.061

0.095

0.066

0.074 

 

Test item treated killed tissues:

5-(Hydroxymethyl)furfural

1

2

3

mean

0.145

0.099

0.111

0.118

OD values and NSC % of additional control:

 Additional colour control  Optical Density (OD)     Non Specific Colour % (NSCliving %)

 5-(Hydroxymethyl)furfural

(test item treated tissues without MTT

incubation)

1

2

mean

0.037

0.043

0.040 

4.8 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
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 utilised testing conditions. The test item 5-(Hydroxymethyl)furfural is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

EpiSkinTMSM test of 5-(Hydroxymethyl)furfural 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. 439,28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2,=95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % 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 (brown), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

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.

The test item is a possible MTT-reducer and has an intrinsic colour (brown). To avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed.

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.As the NSClivingis below 5%, true MTT metabolic conversion for colour interference is not undertaken.

In thisin vitroskin irritation test using the EPISKIN model, the test item 5-(Hydroxymethyl)furfural did not show significantly reduced cell viability in comparison to the negative control (mean corrected value for possible MTT reduction: 72 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected OD and cell viability values within acceptable limits and standard deviation of all calculated viability values (positive and negative control, test item) was below 18. The experiment was considered to be valid.

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 5-(Hydroxymethyl)furfural is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).