Palladium

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Oct 2020-18 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 10 February 2019

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6 July 2012

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Palladium dihydroxide

EC Number:

235-219-2

EC Name:

Palladium dihydroxide

Cas Number:

12135-22-7

Molecular formula:

H2O2Pd

IUPAC Name:

palladium(2+) dihydroxide

Details on test material:

Brown solid, purity 65.3% (as Palladium), stored at room temperature in the dark

In vitro test system

Test system:

human skin model

Remarks:

The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Source species:

human

Cell type:

other: EpiDerm™ tissue consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was crushed and ground in a mortar and with a pestle to improve the consistency. The test item was tested neat.

Details on test system:

Epi-200-SIT kits and MTT-100 assays are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ∅).
EpiDerm™ tissues are shipped with cool packs on medium-supplemented agarose gels in a 24-well plate. Including time in transit, tissues may be stored at 4 °C for up to 6 days prior to use.

Pre-Experiment
Test items which might absorb light in the same range as formazan dye (naturally or after treatment) and test items which might be able to directly reduce the vital dye MTT (to MTT formazan) may interfere with the tissue viability measurements and need the use of additional controls for corrections. Therefore, two pre-experiments were performed to determine the color interference and the MTT interference.

Option for the Main Experiment
Since the test item/ water or test item/ isopropanol solutions changed color significantly in the first pre-experiment and interfered with MTT in the second pre-experiment, the main experiment had to be performed with additional viable, freeze killed and an additional third set of controls in order to avoid double correction. These additional tissues were designated Non-Specific Killed Control (NSKC) and run in duplicates. They are freeze-killed tissues which were treated with the test item but incubated in medium without MTT solution in the MTT assay. At the end Data Correction Procedure III was performed.

Main Experiment
1 Pre-warming of EpiDerm™ Tissues
The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed, and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze and prior to the exposure of the test item and of the controls the EpiDerm™ tissues was inspected for quality:
It was taken care, that
• air bubbles between agarose and insert were not > 30% of the total surface,
• liquid on top of the insert was removed with sterile cotton tips,
• if again moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models was discarded.
The inserts with the EpiDerm™ tissues were transferred into 6-well-plates containing 0.9 ml assay medium and incubated for 60 minutes under standard conditions. Afterwards, the medium was changed and a further pre-incubation for 17 - 23 hours at standard incubation conditions follows.

2 Treatment
After pre-warming of the EpiDerm™ tissues was completed, the negative and positive control, and the test item was added atop the tissues. The test item respectively controls were tested in triplicate tissues with an exposure time of 60 minutes. Within this period the 6-well plates were placed in the incubator for 35 minutes at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
The test item was crushed and ground in a mortar and a pestle to improve the consistency. 25 mg (39.7 mg/cm2 according to guideline) of the test item was applied onto the surface of the tissue. The tissues were wetted with 25 μL DPBS prior to application.
At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. Afterwards, the tissues were incubated in 0.9 mL of fresh assay medium for 24 hours. After this incubation period the medium was changed, and the tissues were incubated for another 18 ± 2 hours at standard incubation conditions. The complete incubation time was about 42 hours.

MTT-Assay
24-well plates were prepared with 300 μl MTT solution per well kept under standard conditions until required.
After the incubation period was completed, the tissues were transferred to the MTT-plates. After a 3 hour ± 5 minutes incubation period under standard conditions the tissues were rinsed with PBS and carefully dried with blotting paper.
The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted within 2-4 hours while shaking at room temperature.
At the end of the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert were discarded.
Per each tissue, 3 × 200 μL aliquots of the formazan solution were transferred into a 96-well, flat bottom, microtiter plate. 200 μL of isopropanol were added to the wells designated as blanks for 96-well plate.

Measurement
The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 μL of positive control or negative control, or25 mg test item were applied on triplicate tissues.

Duration of treatment / exposure:

The test item respectively controls were tested with an exposure time of 60 minutes.

Number of replicates:

The test item respectively controls were tested in triplicate tissues .

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

78.94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study according to OECD439 and under the experimental conditions reported, Palladium dihydroxide is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study in accordance with OECD439 was performed to assess the irritation potential of Palladium dihydroxide by means of the Human Skin Model Test with EpiDermTM tissue models.
The test item proved to be a MTT reducer in the MTT interference pre-experiment. Also, it dyed deionised water (pre-test for colour interference). Therefore, additional tests with freeze-killed tissues, viable tissues (without MTT addition) and non-specific killed controls (NSKC) had to be performed to determine correction factors for calculating the true viability in the main experiment.
Triplicate tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.
After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.
After exposure of the tissues to Palladium dihydroxide the mean relative viability value was 78.94% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
In conclusion, it can be stated that in this study and under the experimental conditions reported, Palladium dihydroxide is non-irritant to skin.