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Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4-5 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
14 June 2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
Official Journal of the European Union No.
L142, dated May 31st, 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM Database Service on Alternative Methods to Animal Experimentation, INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test”
Version / remarks:
Updated December 2011 / February 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(R*,R*)-1,4-dimercaptobutane-2,3-diol
EC Number:
222-468-7
EC Name:
(R*,R*)-1,4-dimercaptobutane-2,3-diol
Cas Number:
3483-12-3
Molecular formula:
C4H10O2S2
IUPAC Name:
1,4-disulfanylbutane-2,3-diol
Specific details on test material used for the study:
Batch-No.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

In vitro test system

Test system:
human skin model
Remarks:
EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN Laboratories Lyon, France
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Remarks:
100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: EPISKIN Laboratories, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 20-EKIN-010
- Expiry date: 09 March 2020
- Date of initiation of testing: 4 May 2020

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.

APPLICATION / EXPOSURE
- Test Item: An amount of 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Positive and negative control: A volume of 50 µL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Additional controls for MTT direct interacting chemicals: In addition to the normal procedure, 2 killed test item treated tissues and 2 killed negative control treated tissues were used for the MTT evaluation in one run in each exposure time (4h, 1h, 3min).
- Exposure: The plates with the treated epidermis units were incubated for the exposure time of 4 h ours at room temperature (22.7-23.1°C). Additional controls for MTT evaluation were also tested with each exposure time (4h, 1h, 3min).

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Duplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 ml PBS 1x Solution, twice (the one extra rinsing was made based on the above way). 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 TEST:
After the exposure of test item was terminated by rinsing with PBS, 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 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION:
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 epidermis material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS:
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 (Linearity range: 0.2720 – 3.4218) using acidified isopropanol solution as the blank (6×200 µL).
Linear OD range of the spectrometer: not reported

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (SubCat.1A) if mean tissue viability is < 35 % after 3 min exposure
- The test substance is considered to be corrosive to skin (subCat.1B or 1C) if Mean tissue viability is = 35 % after 3 min exposure and < 35 % after 1 hour exposure OR Mean tissue viability is = 35 % after 1 hour exposure and < 35 % after 4 hours exposure
- The test substance is considered to be non-corrosive to skin if mean tissue viability is = 35 % after 4 hours exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 mg

VEHICLE
- no, but 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): NaCl 9 g/L

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
4h, 1h and 3min for test item and negative control
4h for the positive control
Duration of post-treatment incubation (if applicable):
no post-treatment incubation
Number of replicates:
Two replicates were used for the test item and control(s) respectively.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
4h exposure
Run / experiment:
1
Value:
57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
difference of viability between the two tissue replicates: 23.5
Positive controls validity:
valid
Remarks:
2% viability, difference of viability between the two tissue replicates: 0.9%
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
1h exposure time
Run / experiment:
1
Value:
68
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability, difference of viability 22%
Positive controls validity:
not examined
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 min exposure
Run / experiment:
1
Value:
77
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability, difference of viability 7.3%
Positive controls validity:
not examined
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 31.61 %, 34.76 % and 22.36 % at the 4h, 1h and 3 min exposure respectively. As the NSMTT were below 50 % in each case the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. Furthermore, the test item was completely removed from the epidermal surface at rinsing per iod. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
-Acceptance criteria met for negative control (4h exposure): yes, OD value 0.791, difference of viability between the two tissue replicates: 23.4% (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 = 30)
- Acceptance criteria met for positive control (4h exposure): yes, 0.016 % viability, difference of viability between the two tissue replicates: 0.9% (The acceptable mean percentage viability range for positive controls is 0-20% and the standard deviation value (SD) of the % viability should be < or = 30.)
- Acceptance criteria met for variability between replicate measurements (4h exposure): yes, difference of viability between the two tissue replicates test item: 18 % (should be < or = 30)

Any other information on results incl. tables

OD values and cell viability percentages of the positive and negative control

 Controls

 Optical Density (OD)

   Viability (%)  Delta%

Negative control

NaCl (9g/L saline)

4h exposure

1

2

mean

SD

CV 

0.791

1.000

0.895

0.148

16.518 

88

112

100

16.518

16.518 

 23.4

Negative control

NaCl (9g/L saline)

1h exposure

1

2

mean

SD

CV 

1.005

0.806

0.905

0.141

15.566 

111

89

100

15.566

15.566 

22.0

 

Negative control

NaCl (9g/L saline)

3 min exposure

1

2

mean

SD

CV

0.918

0.988

0.953

0.049

5.157 

96

104

100

5.157  

5.157

7.3

 

Positive Control:
Glacial acetic acid
4 h exposure 

1

2

mean

SD

CV 

0.012

0.020

0.016

0.005

34.754 

1

2

2

0.608

34.754 

0.9

 

Remark: delta%: The difference of viability between the two relating tissues.

Mean blank OD values were 0.0386 each plate

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

OD Values and viability percentages of the test item

Test Item

 Optical Density (OD)

   TODTT   Viability (%) Delta%   Relative viability (%)

1,4-Dithiothreitol

4h exposure

1

2

mean

SD

CV 

0.916

0.755

0.835

0.114

13.654 

0.552

0.471

0.512

0.057

11.137

102

84

93

12.742

13.654

18.0

 

62

53

57

6.371

11.137 

  

1,4-Dithiothreitol

1h exposure

 

1

2

mean

SD

CV

0.905

0.944

0.924

0.027

2.956 

0.610

0.629

0.619

0.014

2.206 

100

104

102

3.019

2.956 

4.3

 

67

69

68

1.509

2.206 

  

1,4-Dithiothreitol

3 min exposure

1

2

mean

SD

CV 

0.845

0.983

0.914

0.098

10.720 

0.701

0.770

0.736

0.049

6.660 

89

103

96

10.284

10.720 

14.5

 

74

81

77

5.142

6.660 

 

Delta%: The difference of viability between the two relating tissues

TODTT: true MTT metabolic conversion

Mean blank OD values were 0.0386 each plate

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

OD values of additional controls for MTT-interacting test item

 Controls Optical Density (OD)     NSMTT % 

Negativ control killed tissues:

NaCl (9 g/L saline)

4h exposure

1

2

mean

SD

CV 

0.129

0.125

0.127

0.003

2.532 

   31.61

Test Item treated killed tissues:

1,4-Dithiothreitol

4h exposure

1

2

mean

SD

CV

0.441

0.379

0.410

0.044

10.665 

 

Negativ control killed tissues:

NaCl (9 g/L saline)

1h exposure

 

1

2

mean

SD

CV

0.125

0.129

0.127

0.003

2.113 

34.76   
 

Test Item treated killed tissues:

1,4-Dithiothreitol

1h exposure

 

1

2

mean

SD

CV

0.456

0.427

0.442

0.021

4.681 

 

Negativ control killed tissues:

NaCl (9 g/L saline)

3 min exposure

 

1

2

mean

SD

CV

0.166

0.159

0.162

0.005

2.829 

    22.36
 

Test Item treated killed tissues:

1,4-Dithiothreitol

3 min exposure

 

1

2

mean

SD

CV

0.449

0.301

0.376

0.103

27.547 

NSMTT %: non-specific MTT reduction %

Mean blank OD values were 0.0386 each plate

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:
other: The test substance can be classified as Non-corrosive. However, skin irritation potential cannot be excluded.
Remarks:
The study is used for classification in a weight of evidence approach.
Conclusions:
In this in vitro skin corrosion test in EPISKIN model (OECD 431) with 1,4-Dithiothreitol (CAS-No. 3483-12-3) the results indicate that the test item is not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item 1,4-Dithiothreitol can be classified as Non-corrosive. However, skin irritation potential cannot be excluded. Further studies are needed for classification.
Executive summary:

Disks of EPISKIN (two units / chemical / incubation time) were treated with the test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a = 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively.

The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

Each test item treated tissue viabilities were above 35 % of the mean negative control value after 4 hours, 1 hour and 3 minutes exposure. The average test item treated tissue relative viabilities were 57 % at 4 hours, 68 % at 1 hour and 77 % at 3 minutes of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits.

All assay acceptance criteria were met, the experiment was considered to be valid.

In conclusion, in this in vitro skin corrosion test in EPISKIN model (OECD 431) with 1,4-Dithiothreitol (CAS-No. 3483-12-3) the results indicate that the test item is not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item 1,4-Dithiothreitol can be classified as Non-corrosive.

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