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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February 2018 to 02 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February 2018 to 02 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
No correction for purity of the test material was applied.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: SkinEthic, France
- Tissue lot number: 18-EKIN-009

TEST FOR DIRECT MTT REDUCTION
50 µL of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere for 3 hours and then any colour change was observed. After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
10 µL of the test material was added to 90 μL of deionised water and 10 µL of the test material was added to 90 μL of acidified isopropanol. The mixture was shaken for about 15 minutes at room temperature and then colour was checked (unaided visual assessment). As no coloured solution was detected and the test material had no intrinsic colour, no additional colour controls were needed.

MAIN TEST
PRE-INCUBATION
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was 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 overnight at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.

APPLICATION OF TEST MATERIAL AND CONTROLS
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.
10 µL of test material was applied evenly to each of three test units. The test material was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
50 µL of negative control or positive control was added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.9 - 25.5°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
After 15 minutes incubation time, the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

INCUBATION
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 (± 1 hour) at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

MTT TEST
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5 % CO2 for 3 hours, protected from light, in a > 95 % humidified atmosphere.

FORMAZAN EXTRACTION
At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit using a biopsy punch. The epidermis was separated with the aid of forceps and both parts were placed into a tube containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENT
Following the formazan extraction, 2×200 µL sample from each tube were placed into the wells of a 96-well plate. The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.

ACCEPTABILITY CRITERIA
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- Control OD values should be within the historical control range.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
- Standard deviation value (SD) of the negative control tissues and the identically treated replicates % viability should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0 - 40% and the standard deviation value (SD) of the % viability should be ≤ 18.

INTERPRETATION
- Mean tissue viability % is ≤ 50 % = Category 2 or Category 1
- Mean tissue viability % is > 50 % = Non-Irritant
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 5 % (w/v) SDS solution (in distilled water)
Duration of treatment / exposure:
15 minutes (± 0.5 min) at room temperature (23.9 - 25.5 °C)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour) at 37 °C (5 % CO2, in a > 95 % humidified atmosphere)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
(relative)
Run / experiment:
mean
Value:
100
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
DIRECT MTT REDUCTION AND ASSESSMENT OF COLOUR INTERFERENCE
As no colour change (yellow colour) was observed after three hours of incubation of the test material in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
As the test material was colourless and it had no colouring potential, no additional controls were used for the non-specific OD evaluation.

TEST MATERIAL AND CONTROLS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1.
The OD values for the test material treated skin samples showed 100.0 % relative viability.
The mean OD value of the three negative control tissues was in the recommended range (0.810). Standard deviation of the viability results for negative control samples was 3.5 %.
The positive control treated tissues showed 4.5 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0 %.

QUALITY CRITERIA
The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 2.6 %.
All parameters were within acceptable limits and therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance

Measured

OD

Blank

corrected OD

Mean Blank

corrected OD

Viability

(%RV)

Mean Viability

(%RV)

Negative control

0.889

0.842

0.810

104

100.0

0.844

0.797

98.5

0.836

0.789

97.5

Positive control

0.076

0.029

0.036

3.6

4.5

0.083

0.036

4.4

0.091

0.044

5.5

Test material

0.836

0.789

0.810

97.4

100.0

0.878

0.831

102.6

0.856

0.809

99.9

 Notes:

1.         Mean blank value was 0.047.

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

 

Interpretation of results:
other: Non-irritant according to EU criteria
Conclusions:
Following exposure with the test material the mean cell viability was 100.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.
Executive summary:

The skin irritation potential of the test material was investigated in vitro in accordance with the standardised guidelines OECD 439 and EU Method B.46 in a reconstructed human epidermis model, under GLP conditions.

During the study, disks of EPISKIN™ (SM) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, in a > 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS treated epidermis were used as negative control and Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test material is considered to be irritant to skin.

Following exposure with the test material the mean cell viability was 100.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”
Version / remarks:
Official Journal of the European Union No. L142 (31 May 2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: colourless limpid liquid
- Storage conditions: in darkness at room temperature
Specific details on test material used for the study:
No correction for purity of the test material was applied.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: SkinEthic, France
- Tissue lot number: 18-EKIN-009

TEST FOR DIRECT MTT REDUCTION
50 µL of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere for 3 hours and then any colour change was observed. After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
10 µL of the test material was added to 90 μL of deionised water and 10 µL of the test material was added to 90 μL of acidified isopropanol. The mixture was shaken for about 15 minutes at room temperature and then colour was checked (unaided visual assessment). As no coloured solution was detected and the test material had no intrinsic colour, no additional colour controls were needed.

MAIN TEST
PRE-INCUBATION
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was 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 overnight at 37 °C in an incubator with 5% CO2 in a > 95 % humidified atmosphere.

APPLICATION OF TEST MATERIAL AND CONTROLS
The Assay Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. 50 µL of test material was applied evenly to each of two test units.
50 µL of negative control or positive control were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 4 hours (±10 min) at room temperature (23.9-26.4 °C).

REMOVAL OF TEST MATERIAL AND CONTROLS
After 4 hours incubation time, the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

MTT TEST
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours, protected from light, in a > 95 % humidified atmosphere.

FORMAZAN EXTRACTION
At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit using a biopsy punch. The epidermis was separated with the aid of forceps and both parts were placed into a tube containing 500 µL acidified isopropanol. The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENT
Following the formazan extraction, 2×200 µL sample from each tube were placed into the wells of a 96-well plate. The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- Control OD values should be within the historical control range.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
- The difference of viability between the two tissue replicates should not exceed 30 %.
- The acceptable mean percentage viability range for positive controls is ≤ 20 %.

INTERPRETATION
For 2 disks:
If both disks have mean viability of ≥ 35 % = Non Corrosive
If both disks have mean viability of < 35 % = Corrosive (at the corresponding incubation period)
For more than 2 disks:
If the mean value is ≥ 35 % and the variability is less than 50 % = Non Corrosive
If the mean value is < 35 % and the variability is less than 50 % = Corrosive
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied:
50 µL
- Concentration:
0.9 % (w/v) NaCl solution

POSITIVE CONTROL
- Amount(s) applied:
50 µL
Duration of treatment / exposure:
4 hours (±10 min) at room temperature (23.9 - 26.4 °C)
Number of replicates:
2

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
(relative)
Run / experiment:
mean
Value:
48.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
DIRECT MTT REDUCTION AND ASSESSMENT OF COLOUR INTERFERENCE
As no colour change (yellow colour) was observed after three hours of incubation of the test material in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
As the test material was colourless and it had no colouring potential, no additional controls were used for the non-specific OD evaluation.

TEST MATERIAL AND CONTROLS
Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.
The mean OD value for the test material treated skin samples showed 48.3 % relative viability.
The mean OD value of the two negative control tissues was in the recommended range (0.675).
The two positive control treated tissues showed 0.5 % viability demonstrating the proper performance of the assay.

QUALITY CRITERIA
The difference of viability between the two test material-treated tissue samples in the MTT assay was 36.2 %. Although higher degree of variability (above the 30 % acceptability limit) was seen, the individual relative viability values of both replicates indicated a negative result (39.5 and 57.0 % relative viability values, well above the threshold of 35 %). Therefore, this fact was considered not to adversely affect the results or interpretation of the study, and the observed value was considered to be acceptable.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance

Measured OD

Blank

corrected OD

Mean Blank

corrected OD

Viability

(%RV)

Mean Viability

(%RV)

Negative control

0.679

0.632

0.675

93.6

100

0.765

0.718

106.4

Positive control

0.052

0.005

0.004

0.8

0.5

0.049

0.002

0.3

Test material

0.314

0.267

0.326

39.5

48.3

0.432

0.385

57

 

Notes:

1.    Mean blank value was 0.047.

2.    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: Non-corrosive according to EU criteria
Conclusions:
Following exposure with the test material, the mean cell viability was 48.3 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive.
Executive summary:

The corrosivity of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 431 in a reconstructed human epidermis model, under GLP conditions.

During the study, disks of EPISKIN™ (SM) were treated with the test material and incubated for 4 hours at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control). For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is < 35 % the test material is considered to be corrosive to skin.

Under the conditions of the study, the mean cell viability for the test material was 48.3% compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive.