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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:
05 March 2018 - 12 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium hexacyanoferrate
EC Number:
237-323-3
EC Name:
Tripotassium hexacyanoferrate
Cas Number:
13746-66-2
Molecular formula:
C6FeN6.3K
IUPAC Name:
tripotassium hexacyanoferrate
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Dark-red crystalline powder
Storage: At room temperature protected from light
Specific details on test material used for the study:
To protect the test item from light glassware was wrapped in tin-foil.

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:
water
Remarks:
5 µL
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number(s): 18-EKIN-010
- Surface: 0.38 cm^2
- Expiration date: 12 March 2018
- The skin was moistened with 5 µL Milli-Q water

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure to the test item: room temperature
- Temperature of incubations(°C): 36.4 - 37.4°C
- Humidity(%): 64 - 85

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: the test substance was checked for possible direct MTT reduction and color interference in a previous skin corrosion test using EpiDerm as a skin model. It was concluded that the test substance did not interfere with the MTT endpoint.
- 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)
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 ≤40% 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:
20.5 to 28.5 mg
Duration of treatment / exposure:
15 ± 0.5 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:
Mean of 3 replicates
Value:
89
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean tissue viability(%): 3.3
Remarks on result:
no indication of irritation
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 4.6.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 3.3% and the SD of the % viability was 0.3
- Acceptance criteria met for variability between replicate measurements: yes, the variability between tissue treated with the test item was 4.4%.

- Since the mean relative tissue viability for potassium ferricyanide was above 50% is considered to be non-irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, potassium ferricyanide showed to be not irritant to the skin (mean tissue viability of 89%). Therefore, the substance does not need to be classified according to GHS and CLP criteria.