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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:
2016-2-24 to 2016-8-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[1,3-bis(2,4,6-trimethylphenyl)-4,5-dimethylimidazol-2-ylidene](2-thienylmethylidene)(tricyclohexylphosphine)ruthenium(II)dichloride
Cas Number:
1190427-50-9
Molecular formula:
C46H65Cl2N2PRuS
IUPAC Name:
[1,3-bis(2,4,6-trimethylphenyl)-4,5-dimethylimidazol-2-ylidene](2-thienylmethylidene)(tricyclohexylphosphine)ruthenium(II)dichloride
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Remarks:
; the study used EpiDerm™ from MatTek
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test used the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and represents the target organ of the species of interest in vitro and closely mimics the biochemical and physiological properties of the upper parts of human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): 23316 (main experiment) and 23339 (NSC evaluation)
- Production date: 2016-2-24 and 2016-6-1
- Date of initiation of testing: 2016-2-23

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 min and room temperature for 25 ± 1 min
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item.
- Observable damage in the tissue due to washing: -
- Modifications to validated SOP: -

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate reader
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm at 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.551 ± 0.125 and 1.57 ± 0.057
- Barrier function: 7.25 h and 6.51 h
- Contamination: Sterile and Sterile


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Viable tissues
- N. of replicates: 2
- Method of calculation used: Interference [%] = [OD(additional test item treated living tissues without MTT staining)/OD(additional negative control treated living tissue with MTT staining)]*100

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement):
-as recommended in TG 439: The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”) if the tissue viability after exposure and post-incubation is less or equal to 50%.
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): tissues were pre-wetted with 25 μL of sterile DPBS before applying solid test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SDS solution

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mixture of 25 mg test item with 1 ml MTT medium did not result in blue/purple colouring and the test item, consequently, did not directly reduce MTT.

The mixture of 25 mg of the test item with 300 μL isopropanol showed colouring detectable by unaided eye-assessment and absorbed light in the relevant range.
For quantitative correction of results, the non-specific colour was determined by using additional viable tissues incubated with 25 mg of the test item and not subjected to MTT-staining and tissues treated as the control and subsequently incubated with MTT. The non-specific colour was calculated to amount 2.3%, which is well below 5% and, therefore, no correction of the determined tissue viability was necessary.

Any other information on results incl. tables

Please see attached file for results.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50% (99 ± 16%). The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

A valid test for skin irritation according to OECD Guideline and under GLP was carried out with the test substance and did not identify any signs of skin irritation.

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