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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:
key study
Study period:
06 Jan 2017 - 02 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014
Deviations:
yes
Remarks:
For the MTT Assay: the formazan salt was extracted in the refrigerator instead of at room temperature. No impact on study outcome. 50µL test item used in MTT reduction/colour intereference AND test substance exposure, rather than 30µL
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methyldecan-1-al
EC Number:
242-745-6
EC Name:
2-methyldecan-1-al
Cas Number:
19009-56-4
Molecular formula:
C11H22O
IUPAC Name:
2-methyldecanal
Test material form:
liquid
Details on test material:
Appearance: clear, colourless liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epi-200 kits and MTT-100 assays purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes, cultured to form a multilayered, highly differentiated model of the human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.
- Quality control for skin discs: Robustness of the test system (or rather of the test kit) was demonstrated following treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsed 20 times
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: N/A

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm
- Filter: no reference filter
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Two

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin as follows: < 50% mean tissue viability after 3 minutes exposure and < 15% after 60 minutes exposure results in a prediciton of corrosivity.
- The test substance is considered to be non-corrosive to skin as follows: ≥ 50% mean tissue viability after 3 minutes exposure and ≥ 15% after 60 minutes exposure results in a predition of non-corrosivity.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: N/A
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): N/A

VEHICLE - N/A


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): N/A
Duration of treatment / exposure:
(Test item, negative and positive controls): 3 ± 0.5 minutes, 60 ± 5 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Two

Test system

Controls:
yes, concurrent positive control
yes, concurrent negative control
Details on study design:
TEST SITE
- Area of exposure: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).
- % coverage: Not specified
- Type of wrap if used: N/A

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a multipipette containing PBS to remove any residual test material (20 times).
- Time after start of exposure: ('At the end of exposure', no time specified)

OBSERVATION TIME POINTS
3 ± 0.5 minutes, 60 ± 5 minutes

SCORING SYSTEM:
- Method of calculation:
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean OD test item/positive control)/ (mean OD negative )]x 100

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
109.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
107.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: N/A
- Direct-MTT reduction: The colourless test item reduced MTT
Test for Direct MTT Reduction and Colour Interference:
Step 1
50 µL of the test item was added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue and an additional test on viable tissues (without MTT addition) should be performed (step 2). Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item was added to 1 ml of a MTT/DMEM solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. Since the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item proved to reduce MTT and an additional test on freeze-killed tissues (step 4) was performed.
Step 4:
The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item similar to viable tissues.
The test item was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

Data correction procedure:
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)

- Colour interference with MTT: The colourless test item did not change colour when mixed with deionised water.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
- Acceptance criteria met for positive control: the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
- Acceptance criteria met for variability between replicate measurements: Yes
- Mean of historical values if different from the ones specified in the test guideline:
3 minutes, positive control:
Mean viability (%)= 21.60
CV (%): 9.52
Range of viabilities (%): 4.60-39.83

3 minutes, negative control:
Mean OD: 1.68
CV (OD): 4.87
Range of ODs: 1.34-1.93

60 minutes, positive control:
Mean viability (%)= 7.02
CV (%): 14.72
Range of viabilities (%): 2.80-14.77

60 minutes, negative control:
Mean OD: 1.65
CV (OD): 3.19
Range of ODs: 1.32-1.85

Any other information on results incl. tables

Table 1 details the results from all tests performed. Table 2 details the corrosivity classification based on relative mean viability.

Table 1. Results after treatment with test item and controls

DOSE GROUP

EXPOSURE INTERVAL
[MIN]

AB-SORBANCE (OD) 570 NM
WELL 1

AB-SORBANCE (OD) 570 NM
WELL 2

AB-SORBANCE (OD) 570 NM
WELL 3

MEAN AB-SORBANCE (OD) OF 3 WELLS MINUS BLANK

MEAN AB-SORBANCE (OD) OF 2 TISSUES

REL. ABSORBANCE
[% OF NEGATIVE CONTROL]*

MEAN REL. ABSORBANCE
[% OF NEGATIVE CONTROL]*

CV [%]

CORRECTED
ODTEST ITEM**

CORRECTED REL. ABSORBANCE [% OF NEGATIVE CONTROL]***

Blank

 

0.046

0.041

0.035

0.000

 

 

Negative Control Tissue 1

3

1.734

1.538

1.694

1.615

1.606

100.5

100

0.8

 

Negative Control Tissue 2

1.659

1.624

1.631

1.597

99.5

Positive Control Tissue 1

0.497

0.479

0.472

0.442

0.473

27.5

29.4

9.2

Positive Control Tissue 2

0.540

0.547

0.546

0.503

31.3

Test Item Tissue 1

1.675

1.646

1.653

1.617

1.723

100.7

107.3

8.7

1.723- (0.164-0.199) = 1.758

109.5

Test Item Tissue 2

1.864

1.877

1.868

1.829

113.9

Negative ControlFreeze Killed TissueTissue 1

0.232

0.246

0.245

0.200

0.199

 

Negative ControlFreeze Killed TissueTissue 2

0.239

0.238

0.235

0.197

Test ItemFreeze Killed TissueTissue 1

0.201

0.198

0.199

0.159

0.164

Test ItemFreeze Killed TissueTissue 2

0.211

0.210

0.210

0.170

 

 

0.035

0.035

0.034

0.000

 

 

Negative Control Tissue 1

60

1.698

1.625

1.679

1.633

1.622

100.7

100

1.0

 

Negative Control Tissue 2

1.668

1.648

1.617

1.610

99.3

Positive Control Tissue 1

0.158

0.163

0.161

0.126

0.140

7.8

8.7

14.5

Positive Control Tissue 2

0.195

0.192

0.182

0.155

9.5

Test Item Tissue 1

1.753

1.752

1.714

1.705

1.756

105.2

108.3

4.1

1.756- (0.182-0.166) = 1.740

107.3

Test Item Tissue 2

1.840

1.850

1.833

1.807

111.4

Negative ControlFreeze Killed TissueTissue 1

0.234

0.233

0.227

0.191

0.166

 

Negative ControlFreeze Killed TissueTissue 2

0.180

0.181

0.186

0.142

Test ItemFreeze Killed TissueTissue 1

0.219

0.222

0.214

0.178

0.182

Test ItemFreeze Killed TissueTissue 2

0.228

0.227

0.226

0.187

* Relative absorbance (rounded values): ( [100 x (absorbancetest item/positive control)]/ (absorbance negative control ) ) x 100

** ODtest item – (ODtest item freeze-killed – ODnegative control freeze-killed)

*** Corrected relative viability (%) = [mean OD test item - (OD test item freeze killed - OD negative control freeze killed)/mean OD negative control]x 100

Table 2. Classification of corrosivity potential based on relative viability

VIABILITY MEASURED AFTER EXPOSURE TIME POINTS

PREDICTION TO BE CONSIDERED

< 50% after 3 minutes exposure

Corrosive

≥ 50% after 3 minutes exposure AND

< 15% after 60 minutes exposure

Corrosive

≥ 50% after 3 minutes exposureAND

≥ 15% after 60 minutes exposure

Non-corrosive

TEST ITEM IDENTIFIED AS CORROSIVE

< 25% after 3 minutes exposure

Optional Sub-category 1A*

≥ 25% after 3 minutes exposure

A combination of optional Sub-categories 1B and 1C

*According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Sub-category 1A results of the EpiDERMTM test method may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered to be non-corrosive to skin since the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%.



Executive summary:

Skin corrosion potential was measured using the Human Skin Model Test with EpiDerm™tissues models, in accordance with OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (Updated Guideline adopted July 29, 2016) and the MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Due to the test item’s MTT-reducing property, an additional test with freeze-killed tissues to determine a correction factor for true viability calculation was performed.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and subsequently 1 hour.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 20.5 hours in the refrigerator.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance compared to the negative control, both for the 3 minutes exposure period (29.4%) and for the 1 hour exposure period (8.7%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item, the corrected relative absorbance values were not reduced (109.4% after 3 minutes exposure, 107.3% after 1 hour exposure). The threshold for corrosivity defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure was not affected. Therefore, the test item is not considered to be corrosive under experimental conditions, in accordance with EU CLP and UN GHS.

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