Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2017 - 14 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
20 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 3 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Physical appearance: colourless to pale yellow liquid
Storage conditions: at room temperature protected from light, container flushed with nitrogen.
Specific details on test material used for the study:
No correction factor for purity required.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number(s): 17-EKIN-032
- Surface: 0.38 cm^2
- Expiration date: 14 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.6 - 37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with PBS (1 washing step)
- Observable damage in the tissue due to washing: no

- Before the start of the study, the test item was checked for possible direct MTT reduction and color interference. It was concluded that the test item did not interfere with the MTT endpoint.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE:
- Incubation time: 3 hours
- Measurement method: TECAN Infinite M200 Pro Plate Reader (570 nm)
- MTT concentration: 0.3 mg/mL

NUMBER OF REPLICATE TISSUES: 3 for the test substance, the negative and the positive control each.

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

CELL VIABILITY MEASUREMENT: After incubation, tissues were dried and incubated with 2 mL MTT-solution for 3 hours. After incubation, epidermis was separated from the collagen matrix and both parts were extracted with 500 μL isopropanol. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (see table 1 in 'Any other information on materials and methods')
A test item 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 item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item 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 item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 μL

NEGATIVE CONTROL
- Amount applied: 25 μL

POSITIVE CONTROL
- Amount applied: 25 μL, re-spread afer 7 minutes of contact time
Duration of treatment / exposure:
15 +/- 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours; + 3 hours with MTT
Number of replicates:
3 for the test item, the negative control and the positive control each.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates
Value:
12
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
Mean tissue viability: 4.8%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the SD of the % viability was 1.3%.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 4.8% and the SD of the % viability was 2.3%
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviation of the three tissue replicates treated with test substance was 6.3%.

- Since the mean relative tissue viability for the test item was below 50% the test item is considered to be irritant.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Classified as Category 2 based on Regulation (EC) 1272/2008.
Conclusions:
In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, Florantone T showed to be irritant with a tissue viability of 12% after 15 minutes of treatment. Based on these results, the substance is classified as Category 2 (irritant) according to GHS and Regulation (EC) 1272/2008.