Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March 2016 - 21 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP conditions

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number: 16-EKIN-011
- Production / shipping / delivery date: No data
- Date of initiation of testing: 14 March 2016

PRE-TEST PROCEDURE:
- Pre-incubation: On day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C.
- Test item colour interference: To assess colour interference, 10 μL of Peomosa (mono-constituent) was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure / post-treatment incubation: 36.3 - 37.3 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: No data
- Linear OD range of spectrophotometer: No data

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 25 μL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability compared to the negative control tissues (100%)
Value:
74
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD570 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD570

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.066

1.047

0.022

100

1.052

1.024

Positive Control Item

0.398

0.462

0.078

37

44

0.549

52

0.437

43

Test Item

0.796

0.770

0.163

75

74

0.595

57

0.919

90

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100%.

Applicant's summary and conclusion

Interpretation of results:
other: not skin irritating
Remarks:
According to EU CLP 1272/2008 and its amendments.
Conclusions:
Under the conditons of this test, the relative mean tissue viability for the test item determined to be 74%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and its amendments.
Executive summary:

The possible skin irritation potential of Peomosa was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 44% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 74%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC)

and its amendments

.