Palladium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jan - 9 Feb 2021

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:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

updated 06 July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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

Specific details on test material used for the study:

Purity: 97.4% (73.8% palladium), 2.6% water

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Mice, CBA/CaOlaHsd, Recognised as the recommended test system. Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands

Sex:

female

Details on test animals and environmental conditions:

Number of animals for the pre-test: 2 females
Number of animals for the main study: 20 females
Number of animals per group: 5 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): 7 - 8 weeks (pre-test) 8 - 12 weeks (main experiment)
The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by fur clippings. In the pre-experiment, animals were individually marked.

Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry:
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C relative humidity approx. 45-65% (except deviation) artificial light 6.00 a.m. - 6.00 p.m. ventilation: at least eight air changes per hour. NOTE: The relative humidity in the animal room was between approximately 15-65% instead of 45-65% for several days.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

For the main study, dose concentrations of 5, 10 and 25% were selected (the highest concentration tested was the highest concentration that could technically be achieved).

No. of animals per dose:

Number of animals per group: 5 females (nulliparous and non-pregnant)

Details on study design:

Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% suspension in propylene glycol. Grinding of the test item in an agate mortar was used to formulate the test item.
To determine the highest, non-irritant, test concentration that did not induce signs of systemic toxicity, a pre-test was performed. Two mice were treated, by topical (epidermal) application of 10 or 25% concentrations of the test item to the dorsal surface of each ear, once daily on three consecutive days. Body weights were recorded prior to the first application of the test item and before sacrifice. Clinical signs were recorded at least once daily. Any signs of local irritation were documented and a score was used to grade possible erythema of the ear skin. In addition, ear thickness was measured using a micrometer prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2), these samples were immediately pooled (for both animals) and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
The results from the pretest are shown in Appendix 1. Erythema of the ear skin (Score 1), erythema of the scalp, hunched back and wet fur were observed in both mice after dose administration. A concentration of 25% was therefore considered to be the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation.
For the main study, dose concentrations of 5, 10 and 25% were selected (the highest concentration tested was the highest concentration that could technically be achieved).

Test Item Preparation
The test item was placed into an appropriate container on a tared balance and propylene glycol was added (weight per weight). The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly before each dosing occasion.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10 and 25% in propylene glycol. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.9 μCi of 3H-methyl thymidine (equivalent to 79.6 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for approximately 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Observations
Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
Determination of ear weights
In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were recorded manually. The results are shown in Appendix 1.
Determination of Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2020. The SI of α-Hexylcinnamaldehyde in acetone:olive oil (4:1 v/v)) were (ICCR study number 2132700):
0% - SI 1.00
5% - SI 1.70
10% - SI 3.06
25% - SI 7.91
Calculated EC3 value = 9.8%
The SI at 25% is within the historical control range (Oct 2015 - April 2020) of SI 5.59 - 17.6 (n=10).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Variability:

mean DPM per animal (2 lymph nodes)
Vehicle Control Group (propylene glycol): 1654,7 (SD 653,7)
5% Palladium dihydroxide: 1668,1 (SD 284,8)

Test group / Remarks:

5% Pd dihydroxide

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Remarks:

Stimulation Indices of 1.0, 1.8, and 2.5 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol. Since these values were all below the threshold value of 3, the EC3 value could not be calculated and the test item is considered to lack the potential to cause skin sensitisation.

Results 2

Parameter:

SI

Value:

1.8

Variability:

mean DPM per animal (2 lymph nodes)
Vehicle Control Group (propylene glycol): 1654,7 (SD 653,7)
10% Palladium dihydroxide: 2997,5 (SD 1118,8)

Test group / Remarks:

10% Pd dihydroxide

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Remarks:

Stimulation Indices of 1.0, 1.8, and 2.5 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol. Since these values were all below the threshold value of 3, the EC3 value could not be calculated and the test item is considered to lack the potential to cause skin sensitisation.

Results 3

Parameter:

SI

Value:

2.5

Variability:

mean DPM per animal (2 lymph nodes)
Vehicle Control Group (propylene glycol): 1654,7 (SD 653,7)
25% Palladium dihydroxide : 4098,1 (SD 1717,4)

Test group / Remarks:

25% Pd dihydroxide

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Remarks:

Stimulation Indices of 1.0, 1.8, and 2.5 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol. Since these values were all below the threshold value of 3, the EC3 value could not be calculated and the test item is considered to lack the potential to cause skin sensitisation.

Cellular proliferation data / Observations:

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No signs of systemic toxicity were observed during the study period. Slight erythema of the ear skin (Score 1 to 2), erythema of the scalp and wet fur was observed.

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.0, 1.8, and 2.5 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol. Since these values were all below the threshold value of 3, the EC3 value could not be calculated and the test item is considered to lack the potential to cause skin sensitisation.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Palladium dihydroxide was not a skin sensitiser when tested in the LLNA (according to OECD429) up to the limit of solubility.

Executive summary:

Palladium dihydroxide was assessed for its skin sensitising potential in mice, using the Local Lymph Node Assay (LLNA - according to OECD429).
Following a solubility assessment and preliminary screening test, 25% Palladium dihydroxide in propylene glycol was the highest concentration that could technically be achieved and which did not cause excessive skin irritation or clinical signs of toxicity. Therefore palladium dihydroxide was formulated in propylene glycol at concentrations of 5, 10 and 25%, for the LLNA. A control group received the vehicle alone.
Three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10 and 25% in propylene glycol by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved. A control group of five mice was treated with the vehicle (propylene glycol) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter as disintegrations per minute (DPM). The mean DPM value for each test group was divided by the mean DPM for the control group to provide the stimulation index (SI) value for each test group.
The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. Slight erythema of the ear skin (Score 1 to 2), erythema of the scalp and wet fur was observed in all treated mice following administration of the test item on days 1, 2 and 3.
In this study Stimulation Indices (S.I.) of 1.0, 1.8, and 2.5 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol, respectively. Since these values were all below the threshold value of 3, the test item is considered to lack the potential to cause skin sensitisation.
Palladium dihydroxide was not a skin sensitiser when tested in the LLNA up to the limit of solubility.