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Toxicological information

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:
04 - 06 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in a closed container, protected from sunlight

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for irritation testing. It is used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and non-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number(s): 15-EKIN-005
- Date of initiation of testing: 04 Feb 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume of washing steps: rinsed thoroughly with PBS 1x solution
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h (± 5 min)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 21.9 ± 0.4
- Barrier function: 2.2 mg/mL
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Method of calculation used:
Blank: The mean of the 6 blank OD values is calculated
Negative control:
- Individual negative control OD values are corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNC raw – OD blank mean
- The corrected mean OD of the 3 negative control values is calculated: this corresponds to 100% viability
Positive control:
- Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPC raw – OD blank mean
- The corrected mean OD of the 3 positive control values is calculated
- The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
- The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item:
- Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTT raw – OD blank mean
- The corrected mean OD of the 3 test item values is calculated
- The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
- The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the mean relative viability after 15 minutes exposure and 42 h post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-corrosive or non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied: 10 μL
- Concentration (if solution): 1x

POSITIVE CONTROL
- Amount(s) applied:10 μL
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of negative control (DPBS)
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of positive control (5% SLS)
Value:
20
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of test item (undiluted)
Value:
12
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: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
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 ≤ 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.
For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

- Acceptance criteria met for negative control: yes, as the mean OD value of the three negative control tissues was 0.850.
- Acceptance criteria met for positive control: yes, as the mean OD value obtained for the positive control was 0.171 and this result corresponds to 20 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: yes, as each calculated standard deviation value (SD) for the % viability was below 18.

Any other information on results incl. tables

Table 1: MTT assay after 3 hours (± 5 min) exposure

Substance

Tissue No.

OD570

OD570(mean)

Relative viability
[%]

Relative viability (mean)

[%]

Standard Deviation of relative viability
[%]

Negative
Control

1

0.854

0.850

100

100

1.67

2

0.862

101

3

0.835

98

Positive
Control

1

0.212

0.171

25

20

6.32

2

0.110

13

3

0.192

23

Test
Item

1

0.054

0.104

6

12

6.17

2

0.158

19

3

0.101

12

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
based on all available data on skin irritation/corrosion (OECD 439 and OECD 431)
Conclusions:
CLP: Irritant, Cat.2