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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:
27 June - 15 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ no application of correction factor on positive control, no demonstration of technical proficiency, 3 min incubation at room temperature instead of at 37°C (OECD 431)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
/ no application of correction factor on positive control, no demonstration of technical proficiency, 3 min incubation at room temperature instead of at 37°C (OECD 431)
Qualifier:
according to
Guideline:
other: EU method B.40 BIS. (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
/ no demonstration of technical proficiency
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbrauchersch utz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200); 00267
Justification for test system used:
The normal, human-derived epidermal keratinocytes (NHEK) attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Kit (Epi-200, MatTek Corporation (Bratislava, Slovakia)
- Tissue lot number: 23345
- Delivery date: 12 June 2016
- Date of initiation of testing: 27 June 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for the 3 ± 0.5 min exposure periods, 37 ± 1.5 °C for the 60 ± 5 min exposure period

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed 20 times with DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1))
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.783 ± 0.027 (Acceptance criteria: 1.0-3.0)
- Barrier function: ET-50 = 6.16 h (Acceptance criteria: 4.77 - 8.72)
- Morphology: functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: no contamination

NUMBER OF REPLICATE TISSUES: duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 2
- Method of calculation used: True viability = Viability of treated tissue – Interference from test chemical = ODtvt – ODkt; ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue
kt = killed tissues
tkt = treated killed tissue
ukt = untreated killed tissue (NC treated tissue)
Since the interference by the test item extract is < 30% of the negative control value, the net OD of the test item extract treated killed control was subtracted from the mean OD of the test item extract treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (optional Sub-category 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (optional Sub-category 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
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: 50 µl
- Concentration: 79.4 μL/cm2

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8 N
Duration of treatment / exposure:
3 ± 0.5 min and 60 ± 5 min
Number of replicates:
2

Test system

Type of coverage:
other: in vitro system
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
mean value of the test item (100%), 3 min exposure
Value:
114
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
mean value of the test item (100%), 60 min exposure
Value:
56.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, as the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.693 and 1.924)
- Acceptance criteria met for positive control: yes, as the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (7.9%)
- Acceptance criteria met for variability between replicate measurements: yes, as the Coefficient of Variation (CV) is in the range of 20 – 100% viability between tissue replicates is ≤ 30% (values between 3.8% and 16.6%)

Any other information on results incl. tables

Table 1: MTT assay after 3 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

1.795

1.775

0.594

0.647

2.276

2.062

1.820

1.717

0.603

0.685

2.019

1.944

1.850

1.693

0.619

0.658

2.002

1.949

OD570(mean)

1.821

1.728

0.606

0.663

2.099

1.985

OD570(mean values* of replicates)

1.737

0.597

2.005

CV (%)

3.8

6.8

4.0

rel. viability (%)

100.0

34.4

114.0**

Blanc (mean) = 0.038

* after blanc correction of single values

** after data correction because of MTT interference

CV = Coefficient of Variation

Table 2: MTT assays after 60 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

1.924

1.845

0.198

0.158

1.232

0.968

1.895

1.715

0.184

0.166

1.181

0.949

1.768

1.728

0.184

0.167

1.190

0.951

OD570(mean)

1.862

1.763

0.189

0.164

1.201

0.956

OD570(mean values* of replicates)

1.776

0.140

1.042

CV (%)

4.0

12.6

16.6

rel. viability (%)

100.0

7.9

56.7**

Blanc (mean) = 0.038

* after blanc correction of single values

** after data correction because of MTT interference

CV = Coefficient of Variation

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Conclusions:
The test item PIPERITONE was non corrosive to skin according to EU CLP and UN GHS
Executive summary:

After exposure of the tissues to the test item the relative absorbance value did not decrease (114.0%) after 3 minutes exposure. After the 1 hour exposure the viability was reduced to 56.7%. The correction factor derived from the additional test with freeze-killed tissues was considered. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.