Ruthenium, dichloro[1,3-dihydro-4,5-dimethyl-1,3-bis(2,4,6-trimethylphenyl)-2H-imidazol-2-ylidene](2-thienylmethylene)(tricyclohexylphosphine)-, (SP-5-41)-

Skin irritation / corrosion

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

Test material information

Constituent 1

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.