Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-02 to 2015-01-29
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended system by the guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermis (RHE Kit)
- Tissue batch number: 14-RHE-024

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing with minimum of 25 mL
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours +/- 5 min

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: well
- Barrier function: checked
- Morphology: well
- Contamination: none
- Reproducibility: well

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : not applicable

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 42 minutes exposure is less or equal to 50%
- The test substance is considered to be non-irritating to skin if the viability after 42 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 µL

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
- Concentration: 5 % aqueous solution
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
63.77
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: not applicable
- Colour interference with MTT: not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1 Results obtained after in RHE assay

Dose
group

Treatment
Interval

Optical
density
Tissue 1

Optical
density
Tissue 2

Optical
density
Tissue 3

Mean

optical
density

Mean relative
viability in %

Standard

deviation (SD)

in %

Negative
Control

42 min

2.445

2.588

2.510

2.515

100.00

2.85

Positive
Control

42 min

0.022

0.021

0.021

0.021

0.83

2.77

Test item

42 min

1.571

1.634

1.606

1.604

63.77

1.97

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study the test item is considered to be not irritating to human skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test according to OECD TG 438. The test consisted of a topical administration of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE™-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer), positive control (5% aqueous solution of sodium dodecyl sulfate) or test item were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 2.515. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 0.83%. Therefore, the study fulfilled the validity criteria.

The tissue viability after treatment with the test item was 63.77 % and, thus, higher than 50 %, i.e. according to UN GHS classification the test item is considered as non-irritant to skin.

In conclusion, under the conditions of this study, the test item is not considered to possess an irritant potential to skin.