Palladium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No reliable in vitro genotoxicity data were identified for palladium. 

Palladium is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'. The genotoxic potential of palladium dihydroxide was tested in three in vitro assays according to OEC471 (reverse mutation assay), OECD476 and OECD487. The three assays concluded on a clear absence of genotoxic/mutagenic potential.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Sept - 9 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997 as corrected in 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

97.4% Pd(OH)2 (2.6% water)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

The bacteria used in the test were obtained from: • British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 • Trinova Biochem GmbH on 27 June 2017.

Metabolic activation:

with and without

Metabolic activation system:

The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4222 and the protein level was adjusted to 20 mg/mL.

The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Exp1:The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Exp2: As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5, 1.5, 5 15, 50, 150, 500 and 1500 μg/plate. Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

The test item was insoluble in sterile distilled water at 25 and 50 mg/mL, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water (Baxter, Batch 19F03BA1A) at a maximum concentration of 25 mg/mL, therefore, this solvent was selected as the vehicle.

The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer, sonication for 15 minutes at room temperature and homogenisation for 5 minutes. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.6%) of the test item.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Remarks:

2-aminoanthracene

Details on test system and experimental conditions:

Top agar was prepared using 0.6% Bacto agar (lot numbers 9105946 expiry date 01/2024 and 9294156 expiry date 08/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 55979 expiry date 11/2020).

Experiment1
Without Metabolic Activation
A 0.2 mL aliquot of the appropriate concentration of test item or solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described above except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.2 mL of the appropriate concentration of test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). However, substantial reductions in revertant colony frequency (below the 0.5 threshold when compared to concurrent vehicle controls) were noted in the absence of metabolic activation (S9-mix) from 500 μg/plate (TA1537) and from 1500 μg/plate to the remaining strains. In the presence of metabolic activation (S9-mix), substantial reductions in revertant colony frequency were noted from 1500 μg/plate to all of the bacterial strains.
A test item precipitate (fine, brown and particulate in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 2:
Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test.
Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), however substantial reductions in revertant colony frequency (below the 0.5 threshold when compared to concurrent vehicle controls) were again noted in the absence of metabolic activation (S9-mix) from 500 μg/plate (TA100 and TA1537) and from 1500 μg/plate (TA1535, TA98 and WP2uvrA). In the presence of metabolic activation (S9-mix), substantial reductions in revertant colony frequency were noted from 500 μg/plate (TA1537) and from 1500 μg/plate (TA1535, TA98, TA100 and WP2uvrA).
A test item precipitate (fine, brown and particulate in appearance) was noted at 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Conclusions:

In a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471, GLP compliant) the test item Palladium dihydroxide did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test Palladium dihydroxide was considered to be non-mutagenic when tested up to the limit of toxicity.

Executive summary:

Palladium dihydroxide was tested in a GLP-compliant AMES test (acc. to OECD471).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with aqueous suspensions of the test item (based on preliminary solubility checks) using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 0.5 to 1500 μg/plate. Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation. 

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method), however substantial reductions in revertant colony frequency were noted for all of the tester strains, initially from 500 and 1500 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively.
Based on the results of Experiment 1, the toxic limit of the test item (1500 μg/plate) was employed as the maximum concentration in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), however substantial reductions in revertant colony frequency were again noted for all of the tester strains, initially from 500 μg/plate in the absence and presence of metabolic activation (S9-mix).
A test item precipitate (fine, brown and particulate in appearance) was noted from 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Palladium dihydroxide did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test Palladium dihydroxide was considered to be non-mutagenic when tested up to the limit of toxicity.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Sept - 10 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

97.4% Pd(OH)2 (2.6% water)

Target gene:

Assessment of the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The Chinese hamster V79 cell line is recognized in the OECD 476 Test Guideline as being a suitable cell line for this test.

Species / strain / cell type:

other: V79 cell line

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fraction used during the course of the study was purchased from Moltox, Lot No. 4222, Expiry 12 March 2022.

The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Main Experiment.

Test concentrations with justification for top dose:

The molecular weight of the test item was 140.43 therefore the maximum proposed dose level in the solubility test was set at 1404.3 μg/mL, the 10 mM limit dose level and a correction for the purity of the test item of 97.4% was applied to the formulations. The test item formed a suspension in MEM suitable for dosing at 14.04 mg/mL and was therefore selected as the solvent. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991).

A dose range of 0, 5.49, 10.97, 21.94, 43.88, 87.77, 175.54, 351.08, 702.15, and 1404.3 μg/mL was used in the preliminary cytotoxicity test. The maximum dose tested was the 10mM limit concentration of 1404.3 μg/mL. A precipitate of the test item was observed at the end of exposure at and above 87.77 μg/mL in both of the exposure groups.

Main experiment:
The dose levels of the test item (4-h -/+S9): 0-5.5-11-22-44-88-176-352 µg Pd(OH)2/L
At the end of the exposure period, precipitate of the test item was observed at and above 88 μg/mL in both the absence and presence of metabolic activation. Therefore, the lowest precipitating dose level was plated for relative survival growth and expression of induced mutants, as recommended by the OECD 476 guideline, and the subsequent dose levels were discarded as they were considered surplus to requirements.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.

Vehicle / solvent:

The molecular weight of the test item was 140.43 therefore the maximum proposed dose level in the solubility test was set at 1404.3 μg/mL, the 10 mM limit dose level and a correction for the purity of the test item of 97.4% was applied to the formulations. The test item formed a suspension in MEM suitable for dosing at 14.04 mg/mL and was therefore selected as the solvent. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: dimethyl benzanthracene (DMBA)

Details on test system and experimental conditions:

Cell Culture
The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Preliminary Cytotoxicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 2 x 106 cells/225 cm2 flask 2 days prior to dosing. This was demonstrated to provide at least 20 x 106 cells available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The concentrations of test item used were 0, 5.49, 10.97, 21.94, 43.88, 87.77, 175.54, 351.08, 702.15, and 1404.3 μg/mL.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level.
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment. The maximum concentration was limited by precipitate.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 2 x 106 cells/225 cm2 flask 2 days before being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with seven test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The concentrations of test item used were 0, 5.5, 11, 22, 44, 88, 176, and 352 μg/mL in both the absence and presence of metabolic activation.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 days then fixed with methanol and stained with Giemsa. Colonies were manually counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth where necessary. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutant frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical solvent control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical solvent control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful. Any additional work to verify an equivocal response will incur extra charges.

Statistics:

When there is no indication of any significant increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test:
A precipitate of the test item was observed at the end of exposure at and above 87.77 μg/mL in both of the exposure groups.
there was no dose-related reduction in the cloning efficiency (CE) in the absence of metabolic activation, and evidence of modest dose-related reductions in the presence of metabolic activation. It should be noted that the reductions in the presence of metabolic activation were only observed at concentrations beyond the onset of precipitate.

Main experiment:
At the end of the exposure period, precipitate of the test item was observed at and above 88 μg/mL in both the absence and presence of metabolic activation. Therefore, the lowest precipitating dose level was plated for relative survival growth and expression of induced mutants, as recommended by the OECD 476 guideline, and the subsequent dose levels were discarded as they were considered surplus to requirements.
There were no marked concentration-related reductions in the relative survival values in the absence of metabolic activation. As was seen in the preliminary toxicity test, modest reductions in relative survival values were observed in the presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentrations in the main test using a dose range that included the lowest precipitating concentration, in both the absence and presence of metabolic activation, as recommended by the OECD 476 Guideline. The mean mutant frequencies for the test item treated cultures were all within the laboratory 95% control limits of historical solvent control data. These results fulfilled the criteria for a clearly negative outcome.

Conclusions:

Palladium dihydroxide did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation and was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Executive summary:

The potential mutagenicity of palladium dihydroxide was assessed on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. 

Chinese hamster (V79) cells were treated with the test item at seven dose levels, in duplicate, together with solvent (MEM medium) and positive controls in the absence and presence of an S9 metabolic activation system. At the end of the treatment period, cultures were plated out to allow growth and expression of induced mutants. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out to determine cloning efficiency and mutant frequency.
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were 0, 5.5, 11, 22, 44 and 88 µg Pd(OH)2/L.

The solvent Minimal Essential Medium (MEM) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.
The positive control treatments, both in the absence and presence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentrations in the main test using a dose range that included the lowest precipitating concentration in both the absence and presence of metabolic activation, as recommended by the OECD 476 Guideline. The mean mutant frequencies for the test item treated cultures were all within the laboratory 95% control limits of historical solvent control data. These results fulfilled the criteria for a clearly negative outcome.

Palladium dihydroxide did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation and was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

23 Sept - 9 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Substance considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997 as corrected in 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

The bacteria used in the test were obtained from: • British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 • Trinova Biochem GmbH on 27 June 2017.

Metabolic activation:

with and without

Metabolic activation system:

The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4222 and the protein level was adjusted to 20 mg/mL.

The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Exp1:The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Exp2: As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5, 1.5, 5 15, 50, 150, 500 and 1500 μg/plate. Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

The test item was insoluble in sterile distilled water at 25 and 50 mg/mL, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water (Baxter, Batch 19F03BA1A) at a maximum concentration of 25 mg/mL, therefore, this solvent was selected as the vehicle.

The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer, sonication for 15 minutes at room temperature and homogenisation for 5 minutes. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.6%) of the test item.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Remarks:

2-aminoanthracene

Details on test system and experimental conditions:

Top agar was prepared using 0.6% Bacto agar (lot numbers 9105946 expiry date 01/2024 and 9294156 expiry date 08/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 55979 expiry date 11/2020).

Experiment1
Without Metabolic Activation
A 0.2 mL aliquot of the appropriate concentration of test item or solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described above except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.2 mL of the appropriate concentration of test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). However, substantial reductions in revertant colony frequency (below the 0.5 threshold when compared to concurrent vehicle controls) were noted in the absence of metabolic activation (S9-mix) from 500 μg/plate (TA1537) and from 1500 μg/plate to the remaining strains. In the presence of metabolic activation (S9-mix), substantial reductions in revertant colony frequency were noted from 1500 μg/plate to all of the bacterial strains.
A test item precipitate (fine, brown and particulate in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 2:
Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test.
Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), however substantial reductions in revertant colony frequency (below the 0.5 threshold when compared to concurrent vehicle controls) were again noted in the absence of metabolic activation (S9-mix) from 500 μg/plate (TA100 and TA1537) and from 1500 μg/plate (TA1535, TA98 and WP2uvrA). In the presence of metabolic activation (S9-mix), substantial reductions in revertant colony frequency were noted from 500 μg/plate (TA1537) and from 1500 μg/plate (TA1535, TA98, TA100 and WP2uvrA).
A test item precipitate (fine, brown and particulate in appearance) was noted at 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Conclusions:

In a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471, GLP compliant) the test item Palladium dihydroxide did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test Palladium dihydroxide was considered to be non-mutagenic when tested up to the limit of toxicity.

Executive summary:

Palladium dihydroxide was tested in a GLP-compliant AMES test (acc. to OECD471).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with aqueous suspensions of the test item (based on preliminary solubility checks) using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 0.5 to 1500 μg/plate. Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation. 

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method), however substantial reductions in revertant colony frequency were noted for all of the tester strains, initially from 500 and 1500 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively.
Based on the results of Experiment 1, the toxic limit of the test item (1500 μg/plate) was employed as the maximum concentration in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), however substantial reductions in revertant colony frequency were again noted for all of the tester strains, initially from 500 μg/plate in the absence and presence of metabolic activation (S9-mix).
A test item precipitate (fine, brown and particulate in appearance) was noted from 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Palladium dihydroxide did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test Palladium dihydroxide was considered to be non-mutagenic when tested up to the limit of toxicity.

The substance is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

25 Sept - 10 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Substance considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Assessment of the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The Chinese hamster V79 cell line is recognized in the OECD 476 Test Guideline as being a suitable cell line for this test.

Species / strain / cell type:

other: V79 cell line

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fraction used during the course of the study was purchased from Moltox, Lot No. 4222, Expiry 12 March 2022.

The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Main Experiment.

Test concentrations with justification for top dose:

The molecular weight of the test item was 140.43 therefore the maximum proposed dose level in the solubility test was set at 1404.3 μg/mL, the 10 mM limit dose level and a correction for the purity of the test item of 97.4% was applied to the formulations. The test item formed a suspension in MEM suitable for dosing at 14.04 mg/mL and was therefore selected as the solvent. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991).

A dose range of 0, 5.49, 10.97, 21.94, 43.88, 87.77, 175.54, 351.08, 702.15, and 1404.3 μg/mL was used in the preliminary cytotoxicity test. The maximum dose tested was the 10mM limit concentration of 1404.3 μg/mL. A precipitate of the test item was observed at the end of exposure at and above 87.77 μg/mL in both of the exposure groups.

Main experiment:
The dose levels of the test item (4-h -/+S9): 0-5.5-11-22-44-88-176-352 µg Pd(OH)2/L
At the end of the exposure period, precipitate of the test item was observed at and above 88 μg/mL in both the absence and presence of metabolic activation. Therefore, the lowest precipitating dose level was plated for relative survival growth and expression of induced mutants, as recommended by the OECD 476 guideline, and the subsequent dose levels were discarded as they were considered surplus to requirements.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.

Vehicle / solvent:

The molecular weight of the test item was 140.43 therefore the maximum proposed dose level in the solubility test was set at 1404.3 μg/mL, the 10 mM limit dose level and a correction for the purity of the test item of 97.4% was applied to the formulations. The test item formed a suspension in MEM suitable for dosing at 14.04 mg/mL and was therefore selected as the solvent. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: dimethyl benzanthracene (DMBA)

Details on test system and experimental conditions:

Cell Culture
The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Preliminary Cytotoxicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 2 x 106 cells/225 cm2 flask 2 days prior to dosing. This was demonstrated to provide at least 20 x 106 cells available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The concentrations of test item used were 0, 5.49, 10.97, 21.94, 43.88, 87.77, 175.54, 351.08, 702.15, and 1404.3 μg/mL.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level.
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment. The maximum concentration was limited by precipitate.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 2 x 106 cells/225 cm2 flask 2 days before being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with seven test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The concentrations of test item used were 0, 5.5, 11, 22, 44, 88, 176, and 352 μg/mL in both the absence and presence of metabolic activation.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 days then fixed with methanol and stained with Giemsa. Colonies were manually counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth where necessary. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutant frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical solvent control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical solvent control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful. Any additional work to verify an equivocal response will incur extra charges.

Statistics:

When there is no indication of any significant increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test:
A precipitate of the test item was observed at the end of exposure at and above 87.77 μg/mL in both of the exposure groups.
there was no dose-related reduction in the cloning efficiency (CE) in the absence of metabolic activation, and evidence of modest dose-related reductions in the presence of metabolic activation. It should be noted that the reductions in the presence of metabolic activation were only observed at concentrations beyond the onset of precipitate.

Main experiment:
At the end of the exposure period, precipitate of the test item was observed at and above 88 μg/mL in both the absence and presence of metabolic activation. Therefore, the lowest precipitating dose level was plated for relative survival growth and expression of induced mutants, as recommended by the OECD 476 guideline, and the subsequent dose levels were discarded as they were considered surplus to requirements.
There were no marked concentration-related reductions in the relative survival values in the absence of metabolic activation. As was seen in the preliminary toxicity test, modest reductions in relative survival values were observed in the presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentrations in the main test using a dose range that included the lowest precipitating concentration, in both the absence and presence of metabolic activation, as recommended by the OECD 476 Guideline. The mean mutant frequencies for the test item treated cultures were all within the laboratory 95% control limits of historical solvent control data. These results fulfilled the criteria for a clearly negative outcome.

Conclusions:

Palladium dihydroxide did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation and was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Executive summary:

The potential mutagenicity of palladium dihydroxide was assessed on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. 

Chinese hamster (V79) cells were treated with the test item at seven dose levels, in duplicate, together with solvent (MEM medium) and positive controls in the absence and presence of an S9 metabolic activation system. At the end of the treatment period, cultures were plated out to allow growth and expression of induced mutants. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out to determine cloning efficiency and mutant frequency.
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were 0, 5.5, 11, 22, 44 and 88 µg Pd(OH)2/L.

The solvent Minimal Essential Medium (MEM) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.
The positive control treatments, both in the absence and presence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentrations in the main test using a dose range that included the lowest precipitating concentration in both the absence and presence of metabolic activation, as recommended by the OECD 476 Guideline. The mean mutant frequencies for the test item treated cultures were all within the laboratory 95% control limits of historical solvent control data. These results fulfilled the criteria for a clearly negative outcome.

Palladium dihydroxide did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation and was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

The substance is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct 2020 - 16 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

purity: 97.4% Pd(OH)2 (2.6% water)
Formulated concentrations were adjusted to allow for the stated water/impurity content (2.6%) of the test item

Species / strain / cell type:

other: lymphocytes isolated from whole blood of a non-smoking volunteer (18-35)

Remarks:

The details of the donors used are: -Preliminary Toxicity Test: male, aged 30 years -Main Experiment: male, aged 31 years

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal Enzyme Fraction (Moltox)

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose levels of test item used were 0, 5.48, 10.97, 21.94, 43.88, 87.75, 175.5, 351, 702, and 1404 μg/mL. The maximum dose was the 10 mM limit dose level. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 87.75μg/mL in all three of the exposure groups.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment.

For the Main Experiment, the dose levels of test item used were 0, 2.74, 5.48, 10.97, 21.94, 43.88, 87.75, and 175.5 μg/mL. Precipitate observations were recorded at the beginning and end of the exposure periods.

Vehicle / solvent:

The test item was soluble in MEM medium at 14.04 mg/mL in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Test item preparation
Prior to each experiment, the test item was accurately weighed, dissolved in MEM medium, and serial dilutions prepared. There was no significant change in pH (7.44-7.55) when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (310-325 mOsm).
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

Culture conditions
Duplicate lymphocyte cultures (A and B), (quadruplicate for the vehicle) were established for each dose level, by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 8.3 - 9.4 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.4 - 0.5 mL heparinized whole blood.

Test conditions
1/ 4-Hour Exposure With Metabolic Activation (S9)
After 44 to 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL. After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
2/4-Hour Exposure Without Metabolic Activation (S9)
After 44 to 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
3/24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above is a modification of the suggested cell treatment schedule in the 487 Guideline and is considered to be an acceptable alternative (Whitwell et al., 2019). This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item dose levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate cultures for the test item and quadruplicate cultures for the vehicle controls.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Assessments
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.
Coding
The slides were coded before analysis using a computerized random number generator. Cytokinesis Block Proliferation Index (CBPI): a minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPIt – 1) / (CBPIc – 1)} (with t=test chemical treatment culture and c=vehicle control culture)
Where CBPI = (n° mononucleate cells + '2xn° binucleate cells)+(3xn°multinucleate cells) / (total n° of cells)

Scoring of Micronuclei
The micronucleus frequency in 1000 binucleated cells was analyzed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the vehicle controls). Cells with 1, 2 or more micronuclei were recorded and included in the total. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test system is then considered to be unable to induce chromosome breaks and/or gain or loss.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if considered appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Species / strain:

other: lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three of the exposure groups which was 175.5 μg/mL. Precipitate of test item was noted at and above 87.75 μg/mL in all three of the exposure groups.

The CBPI data for the 4-hour exposure groups in the absence and presence of S9 and for the 24-hour exposure groupconfirm the qualitative observations in that no marked dose-related test item-induced toxicity was observed.

The maximum concentration selected for micronuclei frequency analysis in binucleate cells was the lowest precipitating dose level and was 87.75 μg/mL for all three exposure groups.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating dose level. There were also no statistically significant concentration related increases in any of the three exposure groups when evaluated with a trend test.

Conclusions:

Palladium dihydroxide was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro (test according to OECD487). The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro when tested to its lowest precipitating dose level.

Executive summary:

An in vitro study (according to OECD487) was performed to determine the clastogenic and aneugenic potential of the palladium dihydroxide on the nuclei of normal human lymphocytes.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration, and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate in all three of the exposure groups. The dose levels selected for the Main Experiment were 0, 2.74, 5.48, 10.97, 21.94, 43.88, 87.75, 175.5 µg palladium dihydroxide/mL.

All vehicle (MEM medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item was non-toxic and did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating dose level. There were also no statistically significant concentration related increases in any of the three exposure groups when evaluated with a trend test.

Palladium dihydroxide was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro (test according to OECD487). The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro when tested to its lowest precipitating dose level.

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

20 Oct 2020 - 16 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Substance is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

other: lymphocytes isolated from whole blood of a non-smoking volunteer (18-35)

Remarks:

The details of the donors used are: -Preliminary Toxicity Test: male, aged 30 years -Main Experiment: male, aged 31 years

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal Enzyme Fraction (Moltox)

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose levels of test item used were 0, 5.48, 10.97, 21.94, 43.88, 87.75, 175.5, 351, 702, and 1404 μg/mL. The maximum dose was the 10 mM limit dose level. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 87.75μg/mL in all three of the exposure groups.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment.

For the Main Experiment, the dose levels of test item used were 0, 2.74, 5.48, 10.97, 21.94, 43.88, 87.75, and 175.5 μg/mL. Precipitate observations were recorded at the beginning and end of the exposure periods.

Vehicle / solvent:

The test item was soluble in MEM medium at 14.04 mg/mL in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Test item preparation
Prior to each experiment, the test item was accurately weighed, dissolved in MEM medium, and serial dilutions prepared. There was no significant change in pH (7.44-7.55) when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (310-325 mOsm).
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

Culture conditions
Duplicate lymphocyte cultures (A and B), (quadruplicate for the vehicle) were established for each dose level, by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 8.3 - 9.4 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.4 - 0.5 mL heparinized whole blood.

Test conditions
1/ 4-Hour Exposure With Metabolic Activation (S9)
After 44 to 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL. After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
2/4-Hour Exposure Without Metabolic Activation (S9)
After 44 to 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
3/24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above is a modification of the suggested cell treatment schedule in the 487 Guideline and is considered to be an acceptable alternative (Whitwell et al., 2019). This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item dose levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate cultures for the test item and quadruplicate cultures for the vehicle controls.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Assessments
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.
Coding
The slides were coded before analysis using a computerized random number generator. Cytokinesis Block Proliferation Index (CBPI): a minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPIt – 1) / (CBPIc – 1)} (with t=test chemical treatment culture and c=vehicle control culture)
Where CBPI = (n° mononucleate cells + '2xn° binucleate cells)+(3xn°multinucleate cells) / (total n° of cells)

Scoring of Micronuclei
The micronucleus frequency in 1000 binucleated cells was analyzed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the vehicle controls). Cells with 1, 2 or more micronuclei were recorded and included in the total. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test system is then considered to be unable to induce chromosome breaks and/or gain or loss.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if considered appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Species / strain:

other: lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three of the exposure groups which was 175.5 μg/mL. Precipitate of test item was noted at and above 87.75 μg/mL in all three of the exposure groups.

The CBPI data for the 4-hour exposure groups in the absence and presence of S9 and for the 24-hour exposure groupconfirm the qualitative observations in that no marked dose-related test item-induced toxicity was observed.

The maximum concentration selected for micronuclei frequency analysis in binucleate cells was the lowest precipitating dose level and was 87.75 μg/mL for all three exposure groups.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating dose level. There were also no statistically significant concentration related increases in any of the three exposure groups when evaluated with a trend test.

Conclusions:

Palladium dihydroxide was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro (test according to OECD487). The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro when tested to its lowest precipitating dose level.

Executive summary:

An in vitro study (according to OECD487) was performed to determine the clastogenic and aneugenic potential of the palladium dihydroxide on the nuclei of normal human lymphocytes.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration, and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate in all three of the exposure groups. The dose levels selected for the Main Experiment were 0, 2.74, 5.48, 10.97, 21.94, 43.88, 87.75, 175.5 µg palladium dihydroxide/mL.

All vehicle (MEM medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item was non-toxic and did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating dose level. There were also no statistically significant concentration related increases in any of the three exposure groups when evaluated with a trend test.

Palladium dihydroxide was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro (test according to OECD487). The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro when tested to its lowest precipitating dose level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No reliable in vivo genotoxicity data were identified.

 

However, in a limited dominant lethal mutation assay, no evidence of genotoxicity was seen following a single subcutaneous injection of powdered elemental palladium to male mice prior to mating with untreated females (Arnold et al., 1975).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Brief abstract, limted reporting. Also, the study has several deviations (e.g. inappropriate route, single dose, no positive control) and does not meet the acceptence critiera listed in the current OECD Test Guideline (478)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dominant lethal study in mice to detect mutagenic potential (usually as a result of chromosome aberrations). Treated males were housed with 3 untreated, virgin females/week for 6 consecutive weeks. Mating ability, impregnating ability, viable embryos and percent early deaths were assessed.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: albino

Sex:

male

Details on test animals or test system and environmental conditions:

No details reported in brief abstract.

Route of administration:

subcutaneous

Vehicle:

Saline (suspension)

Details on exposure:

Single subcutaneous injection of 100 mg powdered material as a suspension in saline (1 ml)

Duration of treatment / exposure:

n/a

Frequency of treatment:

Single dose administered

Post exposure period:

[Presumably] 6 weeks

Remarks:

Doses / Concentrations:
100 mg
Basis:
other: nominal in saline

No. of animals per sex per dose:

10 males

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

Dominant lethal mutations were measured as the percentage of all implants that resulted in early death in utero (i.e. percentage resorptions).

Evaluation criteria:

Early (in utero) deaths as a percentage of all implants were compared in treated and control animals

Statistics:

None reported

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Mortality and behavioural assessment only

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Numbers of viable embryos: 10.4-11.7 and 10.2-11.5 in control groups; 11.1-11.9 in treated animals.
Percentage early deaths: 3.0-6.7 and 4.8-8.1 in control groups; 4.2-8.6 in treated group

Observations, viable embryo numbers, percentage of early deaths as a proportion of implantations did not differ between treated and control animals.

Conclusions:

Interpretation of results (migrated information): negative
In a limited dominant lethal mutation assay, no evidence of genotoxicity was seen following a single subcutaneous injection of powdered elemental palladium to male mice prior to mating with untreated females.

Executive summary:

In a limited non-guideline pre-GLP study, elemental palladium was tested for its ability to induce heritable genetic damage (usually indicative of chromosome aberrations) in a dominant lethal mutation assay in albino mice. Single subcutaneous injections of 100 mg powdered palladium in saline (1 ml) were given to 10 males before housing with 3 untreated, virgin females/week for 6 consecutive weeks. The percentage of implants that resulted in early foetal deaths (in utero) did not differ from those measured in saline-treated controls.

In conclusion, a mutagenic effect (indicative of chromosome aberrations) was not indicated for elemental palladium under the conditions of this limited dominant lethal mutation assay in mice.

Its worth noting, that the study has several deviations (e.g. inappropriate route, single dose, no positive control) and does not meet the acceptence criteria listed in the current OECD Test Guideline (478)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

No reliable genotoxicity data for palladium metal were identified.

 

However, in a limited non-guideline pre-GLP study, elemental palladium was tested for its ability to induce heritable genetic damage (usually indicative of chromosome aberrations) in a dominant lethal mutation assay in albino mice. Single subcutaneous injections of 100 mg powdered palladium in saline (1 ml) were administered to 10 males before housing with 3 untreated, virgin females/week for 6 consecutive weeks. The percentage of implants that resulted in early foetal deaths (in utero) did not differ from those measured in saline-treated controls. In conclusion, a mutagenic effect (indicative of chromosome aberrations) was not indicated for elemental palladium under the conditions of this limited dominant lethal mutation assay in mice (Arnold et al., 1975). It is worth noting, that the study has several deviations (e.g. inappropriate route, single dose, no positive control) and does not meet the acceptance criteria listed in the current OECD Test Guideline (478).

 

Further, availability considerations provide good support for the conclusion that genotoxicity testing can be waived. Palladium is considered to be non-bioavailable following oral and dermal exposure, as evidenced by transformation/dissolution (cf. Skeaff 2011, section 4.8) and bio-elution (cf. Rodrigues 2012 a,b,c,d,e, section 7.12) test data. Palladium is not expected to reach the lungs in appreciable quantities (based on respiratory tract deposition modelling data (cf. Selck and Parr, section 4.5). Thus, inhalation will not be a significant route of exposure.

 

Since a chemical is required to be bioavailable in order to induce genotoxicity, palladium is not considered to pose a toxicity hazard for this endpoint. Also, palladium is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'. The genotoxic potential of palladium dihydroxide was tested in three in vitro assays according to OEC471 (reverse mutation assay), OECD476 and OECD487. The three assays concluded on a clear absence of genotoxic/mutagenic potential. Consequently, no further testing for genotoxicity of palladium is considered justified.

Justification for classification or non-classification

No reliable genotoxicity data are available for palladium. However, such effects are not expected, based on a lack of bioavailability following exposure via the oral, dermal and inhalation routes, with support from the limited dominant lethal study in mice. Also, palladium is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'. The genotoxic potential of palladium dihydroxide was tested in three in vitro assays according to OEC471 (reverse mutation assay), OECD476 and OECD487. The three assays concluded on a clear absence of genotoxic/mutagenic potential.

As such, there is no evidence to classify palladium for germ cell mutagenicity according to EU CLP criteria (EC 1272/2008).