Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Palladium dinitrate was negative in good quality, guideline, in vitro studies for genotoxicity. In an OECD Test Guideline 471 study, to GLP, palladium dinitrate dihydrate failed to induce an increase in mutation frequency in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli strain WP2 uvr A, either with or without S9, when tested at up to the limits of cytotoxicity (Verspeek-Rip, 2003).

In an OECD Test Guideline 476 study, to GLP, palladium dinitrate did not induce biologically relevant increases in mutant frequency at the hprt locus of mouse lymphoma (L5178Y) cells when tested up to precipitating or cytotoxic concentrations in the absence and presence of S9 (Lloyd, 2014a).

In an OECD Test Guideline 487 study, to GLP, palladium dinitrate failed to induce biologically significant increases in the frequency of micronuclei in Chinese hamster ovary (CHO) cells when tested up to precipitating or cytotoxic concentrations, both in the presence and absence of rat liver S9 metabolic activation (Lloyd, 2014b).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2002 to 16 December 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD, EU), to GLP, on the dihydrate
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for S. typhimurium strains; tryptophan for E.coli WP2 uvrA
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9, microsomal fraction derived from Aroclor 1254-induced rat liver. The S9 mix contained 5% (v/v) S9 fraction in the first two studies and 10% (v/v) in the two further studies.
Test concentrations with justification for top dose:
Experiment 1: 3, 10, 33, 100, 333, 1000, 3300 and 5000 μg/plate for TA100 and WP2 uvrA.
Experiment 2: 3, 10, 33, 100, 333 and 666 μg/plate for TA15335, TA1537 and TA98.
Experiment 3: 3, 10, 33, 100 and 250 μg/plate for TA15335, TA1537 and TA98. 3, 10, 33, 100, 333 and 666 μg/plate for TA100 and WP2 uvrA.
Experiment 4: 10, 33, 100, 333 and 666 μg/plate for TA1535 and TA98 with S9 only (since not enough cytotoxicity was seen in experiment 3)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: soluble after vortexing
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
650 μg/plate for TA100 without S9
Positive control substance:
sodium azide
Remarks:
5 μg/plate for TA1535 without S9
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 μg/plate for WP2 uvrA without S9
Positive control substance:
9-aminoacridine
Remarks:
60 μg/plate for TA1537 without S9
Positive control substance:
other: daunomycin
Remarks:
4 μg/plate for TA98 without S9
Positive control substance:
other: 2-aminoanthracene
Remarks:
With 5% S9: 1 μg/plate for TA1535, TA98 and TA100; 2.5 μg/plate for TA1537; 5 μg/plate for WP2 uvrA. With 10% S9: 2.5 μg/plate for TA1535, TA1537, TA98 and TA100; 10 μg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hr

NUMBER OF REPLICATIONS: plates prepared in triplicate.
Each strain was tested in two independent studies; a further study was performed with TA1535 and TA98 (with S9 only), since insufficient cytotoxicity was observed in one study where the highest dose was 250 μg/plate.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:

Evaluation criteria:
The test substance was considered to be mutagenic if the number of revertant colonies was at least twice that of the spontaneous revertants and reproducible in at least one independently repeated experiment. However any mean plate count of less than 20 revertants was considered to be not significant.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
yes: TA1537, TA100. no: TA1535, TA98
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535; TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was seen at 3330 and 5000 μg/plate
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes and acceptable minimum and maximum numbers of spontaneous revertants and revertants induced by the positive controls given in the report

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In the study with TA1535 and TA98 with S9, a footnote to the table says the vehicle control used was DMSO, but there is no explanation of why and its use is not mentioned in the text.

Conclusions:
Interpretation of results (migrated information):
negative

In an OECD Test Guideline 471 study, to GLP, palladium dinitrate dihydrate failed to induce an increase in mutation frequency in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli strain WP2 uvr A, either with or without S9, when tested at up to the limits of cytotoxicity.
Executive summary:

The mutagenic potential of palladium dinitrate dihydrate was assessed in a reverse mutagenicity assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assessed in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and in Escherichia coli WP2 uvrA, in an attempt to detect both base-pair substitution and frameshift mutations.

In a study with strains TA100 and WP2 uvrA (which also served as a range-finding study), without or with 5% S9 fraction in the S9 mix, the test substance precipitated at dose levels of 3330 and 5000 μg/plate. Cytotoxicity was seen at concentrations of 1000 μg/plate and above with TA100, and at 333 or 1000 μg/plate without and with S9, respectively, with WP2 uvrA. When tested with TA1535, TA1537 and TA98, without or with 5% S9 fraction in the S9 mix, at concentrations of up to 666 μg/plate cytotoxicity was seen at levels of 333 μg/plate and above. In a study in which the S9 mix contained 10% S9, cytotoxicity was observed in all the test strains, both with and without metabolic activation, apart from TA1535 and TA98 in the presence of S9 only. Since the highest dose used for these latter strains was only 250 μg/plate, a further experiment was performed in which the top dose was 666 μg/plate; toxicity was observed for both strains.

Palladium dinitrate dihydrate did not cause an increase in mutation in these studies, either with or without S9. In contrast, the known mutagens used as positive controls showed the expected mutagenic activity

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt Locus.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 growth media containing L-glutamine and HEPES.
- Properly maintained: no data.
- Periodically checked for Mycoplasma contamination: yes.
- Periodically checked for karyotype stability: no data.
- Periodically "cleansed" against high spontaneous background: no data.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Range-finder: 0, 37.5, 75, 150, 300, 600 and 1200 ug/mL, limited by solubility in culture medium.
Experiment 1: eleven concentrations, ranging from 100 to 2665 ug/mL, without S-9; nine concentrations, ranging from 100 to 1700 ug/mL, with S-9.
Experiment 2: eleven concentrations, ranging from 150 to 2000 ug/mL, without S-9; nine concentrations, ranging from 150 to 1700 ug/mL, with S-9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water.
- Justification for choice of solvent/vehicle: no data.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Preincubation period: not applicable.
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 7 days.
- Selection time (if incubation with a selection agent): not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable.

SELECTION AGENT (mutation assays): not applicable.
SPINDLE INHIBITOR (cytogenetic assays): not applicable.
STAIN (for cytogenetic assays): not applicable.

NUMBER OF REPLICATIONS: each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments).

NUMBER OF CELLS EVALUATED: at least 10^7.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; percentage relative survival (%RS).

OTHER EXAMINATIONS: no data.
Evaluation criteria:
The test article was considered to induce forward mutations at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the vehicle control (p<=0.05)
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<=0.05)
3. The effects described above were reproducible.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment.

Test for linear trend: chi-squared (one-sided).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested up to precipitating and/or toxic concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In Experiments 1 and 2, when tested up to precipitating and/or toxic concentrations, no statistically significant increases in mutant frequency (MF) were observed following palladium dinitrate treatment at any concentration analysed in the absence and presence of S9. A statistically significant linear trend (p≤0.01) was seen in the absence of S9 in Experiment 1. However, as there were no statistically significant increases in MF at any concentration analysed in Experiment 1 and the effect was not reproduced between experiments in the absence of S9, this isolated observation was considered not biologically relevant.


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/Effects of osmolality: no marked changes in osmolality or pH were observed in the range-finder (in the absence and presence of S-9) or Experiment 1 (in the absence of S-9 only). No data on changes in osmolality or pH in Experiment 1 (in the presence of S-9) or Experiment 2.
- Evaporation from medium: no data.
- Water solubility: preliminary solubility test data were considered in selecting concentrations for analysis.
- Precipitation: in the cytotoxicity range-finder experiment, precipitate was observed at the highest concentration tested (1200 ug/mL) in the presence of S-9, at the end of the 3-hour incubation period. In Experiment 1, precipitate was observed at the highest three concentrations tests (1700-2665 ug/mL in the absence of S-9 and 1200-1700 ug/mL in the presence of S-9) after the 3-hour incubation period. In Experiment 2, precipitate was observed at the highest eight concentrations in the absence of S-9 (600-2000 ug/mL) and the highest six concentrations in the presence of S-9 (600-1700 ug/mL), again at the end of the 3-hour incubation period.

RANGE-FINDING/SCREENING STUDIES: in the cytotoxicity range-finder experiment, six concentrations (37.5-1200 ug/mL) were tested in the absence and presence of S-9, limited by the solubility limit in the culture medium.

COMPARISON WITH HISTORICAL CONTROL DATA: historical control data are included in the study report, but are not discussed in the text. Calculated ratios of mutant frequencies (current frequencies/historical frequencies) ranged from 0.484 to 1.736.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In an OECD Test Guideline 476 study, to GLP, palladium dinitrate did not induce biologically relevant increases in mutant frequency at the hprt locus of mouse lymphoma (L5178Y ) cells when tested up to precipitating or cytotoxic concentrations in the absence and presence of S9
Executive summary:

The mammalian cell mutagenicity of palladium dinitrate was evaluated in mouse lymphoma (L5178Y) cells. The study was undertaken in compliance with OECD Test Guideline 476 and according to GLP.

In a cytotoxicity range finding study, six concentrations (37.5 -1200 μg/mL) were tested (with and without S9 metabolic activation); precipitation was seen after 3 hr incubation at the highest concentration with S9, only. Mutagenicity testing was carried out in two experiments, each assessing 11 concentrations without S9 and 9 concentrations with S9.

Precipitation was observed at the end of the 3-hr incubation period in both experiments. In Experiment 1, precipitation was observed above 1700 μg/mL (without S9) and above 1200 μg/mL (with S9). In Experiment 2, precipitation was seen above 600 μg/mL (with and without S9). All testing concentrations giving viable cultures were evaluated for mutagenicity.

In Experiments 1 and 2, when tested up to precipitating and/or toxic concentrations, no statistically significant increases in mutant frequency (MF) were observed following palladium dinitrate treatment at any concentration analysed in the absence and presence of S9. A statistically significant linear trend (p≤0.01) was seen in the absence of S9 in Experiment 1. However, as there were no statistically significant increases in MF at any concentration analysed in Experiment 1 and the effect was not reproduced between experiments in the absence of S9, this isolated observation was considered not biologically relevant.

Overall, palladium dinitrate failed to induce biologically significant increases in mutation frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic/precipitating concentrations in two independent experiments, each in the absence and presence of S9.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: Chromosome damage: micronucleus formation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 February 2014 - 14 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, to GLP, with minor deviations which are not expected to have affected the study validity.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 487: In vitro mammalian cell micronucleus test
Deviations:
yes
Remarks:
Minor protocol deviations relating to pulse treatment duration, test article formulation and (in the range-finder experiment) trypsinisation would not have affected the integrity of the study or the interpretation of its results.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:McCoy's 5A medium including 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: "reconstituted for each experiment so as to maintain karyotypic stability"
- Periodically "cleansed" against high spontaneous background: "subcultured regularly at low density and before overgrowth occurs to maintain low aberration frequencies"
Metabolic activation:
with and without
Metabolic activation system:
S-9 from Aroclor 1254-induced Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder with and without S9 (3+21-hour treatments) and without S9 (24+0-hour treatments):
0, 9.669, 16.11, 26.86, 44.76, 74.60, 124.3, 207.2, 345.4, 575.6, 959.4, 1599, 2665 ug/ml

4+20-hour treatments with and without S9:
0, 200, 400, 600, 800, 1000 (with S9 only), 1200, 1500, 1800 (without S9 only), 2000, 2400, 2665 ug/ml

24+0-hour treatments without S9:
0, 100, 200, 300, 400, 500, 600, 700, 775, 850, 925, 1000, 1200, 1600 ug/ml





Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine
Remarks:
Mitomycin C for 4+20-hours treatment without S9; cyclophosphamide for 4+20-hours treatment with S9; vinblastine for 24+0-hours treatment without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in solution

DURATION: 4-hours (plus 20 hours recovery) with and without S9 or 24 hours (plus 0 hours recovery) without S9

NUMBER OF CELLS EVALUATED: One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei in the main experiment (minimum 200 mono-, bi- and multinucleate per concentration for the range-finder). The number of cells containing micronuclei and the number of micronuclei per cell on each slide were recorded.

DETERMINATION OF CYTOTOXICITY
- Method: replication index (RI) calculation

Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN (micronucleated binucleate) cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.



Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test .
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test.
Probability values of p less-than-or-equal-to 0.05 were accepted as significant.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
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
Positive controls validity:
valid
Additional information on results:
Treatment of cells with palladium dinitrate for 4+20 hr in the absence and presence of S9 and for 24+0 hr in the absence of S9 resulted in frequencies of MNBN cells that were similar to (and not significantly higher than) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequencies of all cultures analysed under all treatment conditions were within the normal ranges.
Remarks on result:
other: other: 24+0-hours treatment
Remarks:
Migrated from field 'Test system'.

Treatment

Concentration (ug/ml)

Cytotoxicity (%)$

Mean MNBN Cell Frequency (%)

Historical Control Range (%)#

Statistical Significance

4+20 hour –S-9

Vehiclea

-

0.50

0.10-1.70

-

 

400.0

3

0.60

 

NS

 

800.0

7

0.70

 

NS

 

1200

7

0.45

 

NS

 

*MMC, 0.08

ND

4.45

 

p≤0.001

4+20 hour +S-9

Vehiclea

-

0.50

0.30-1.80

-

 

600.0

0

0.65

 

NS

 

800.0

0

0.65

 

NS

 

1000

0

0.70

 

NS

 

*CPA, 6.00

ND

11.75

 

p≤0.001

24+0 hour –S-9

Vehiclea

-

0.50

0.10-1.50

-

 

200.0

4

0.45

 

NS

 

400.0

18

0.25

 

NS

 

500

50

0.30

 

NS

 

*VIN, 0.008

ND

7.50

 

p≤0.001

a             Vehicle control was purified water

*             Positive control

#             95thpercentile of the observed range

             Ranges are based om 3+21 hour treatments, therefore used as a guide for 4+20 hour treatments

$             Based on replication index

NS           Not significant

ND           Not determined

Conclusions:
Interpretation of results (migrated information):
negative

In an OECD Test Guideline 487 study, to GLP, palladium dinitrate failed to induce biologically significant increases in the frequency of micronuclei in Chinese hamster ovary (CHO) cells when tested up to precipitating or cytotoxic concentrations, both in the presence and absence of rat liver S9 metabolic activation.
Executive summary:

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 487 (in vitro mammalian cell micronucleus test), palladium dinitrate was tested for its ability to induce chromosome damage in the form of micronuclei.

Chinese hamster ovary (CHO) cells were treated for either 4 hours (with 20-hr growth period) in the presence and absence of rat liver (S9) metabolic activation, or for 24-hr in the absence of S9. Treatments covered a broad range of concentrations, separated by narrow intervals; the frequencies of micronucleated binucleate (MNBN) cells were compared to those observed from vehicle-treated (and positive-)controls for only three concentrations from each regime. The highest concentration in each case was either that inducing precipitation (for the 4 +20-hr treatment, 1200 and 1000 μg/ml in the absence and presence of S9, respectively), or that inducing cytotoxicity (for the 24+0 -hr treatment, 500 μg/ml).

Frequencies of MNBN cells were not significantly higher than those observed in concurrent vehicle controls for any of the concentrations analysed. As a result, palladium dinitrate can be considered non-clastogenic under the conditions of this study, with and without S9

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo data were identified.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No studies conducted in humans or human cells were identified.

 

The mutagenic potential of palladium dinitrate dihydrate was assessed in a reverse mutagenicity assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assessed in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and in Escherichia coli WP2 uvrA, in an attempt to detect both base-pair substitution and frameshift mutations. In a study with strains TA100 and WP2 uvrA (which also served as a range-finding study), without or with 5% S9 fraction in the S9 mix, the test substance precipitated at dose levels of 3330 and 5000 μg/plate. Cytotoxicity was seen at concentrations of 1000 μg/plate and above with TA100, and at 333 or 1000 μg/plate without and with S9, respectively, with WP2 uvrA. When tested with TA1535, TA1537 and TA98, without or with 5% S9 fraction in the S9 mix, at concentrations of up to 666 μg/plate cytotoxicity was seen at levels of 333 μg/plate and above. In a study in which the S9 mix contained 10% S9, cytotoxicity was observed in all the test strains, both with and without metabolic activation, apart from TA1535 and TA98 in the presence of S9 only. Since the highest dose used for these latter strains was only 250 μg/plate, a further experiment was performed in which the top dose was 666 μg/plate; toxicity was observed for both strains. Palladium dinitrate dihydrate did not cause an increase in mutation in these studies, either with or without S9. In contrast, the known mutagens used as positive controls showed the expected mutagenic activity (Verspeek-Rip, 2003).

 

The mammalian cell mutagenicity of palladium dinitrate was evaluated in mouse lymphoma (L5178Y) cells. The study was undertaken in compliance with OECD Test Guideline 476 and according to GLP. In a cytotoxicity range finding study, six concentrations (37.5 - 1200 μg/mL) were tested (with and without S9 metabolic activation); precipitation was seen after 3 hr incubation at the highest concentration with S9, only. Mutagenicity testing was carried out in two experiments, each assessing 11 concentrations without S9 and 9 concentrations with S9. Precipitation was observed at the end of the 3-hr incubation period in both experiments. In Experiment 1, precipitation was observed above 1700 μg/mL (without S9) and above 1200 μg/mL (with S9). In Experiment 2, precipitation was seen above 600 μg/mL (with and without S9). All testing concentrations giving viable cultures were evaluated for mutagenicity. In Experiments 1 and 2, when tested up to precipitating and/or toxic concentrations, no statistically significant increases in mutant frequency (MF) were observed following palladium dinitrate treatment at any concentration analysed in the absence and presence of S9. A statistically significant linear trend (p≤0.01) was seen in the absence of S9 in Experiment 1. However, as there were no statistically significant increases in MF at any concentration analysed in Experiment 1 and the effect was not reproduced between experiments in the absence of S9, this isolated observation was considered not biologically relevant. Overall, palladium dinitrate failed to induce biologically significant increases in mutation frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic/precipitating concentrations in two independent experiments, each in the absence and presence of S9 (Lloyd, 2014a).

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 487 (in vitro mammalian cell micronucleus test), palladium dinitrate was tested for its ability to induce chromosome damage in the form of micronuclei. Chinese hamster ovary (CHO) cells were treated for either 4 hours (with 20-hr growth period) in the presence and absence of rat liver (S9) metabolic activation, or for 24 hr in the absence of S9. Treatments covered a broad range of concentrations, separated by narrow intervals; the frequencies of micronucleated binucleate (MNBN) cells were compared to those observed from vehicle-treated (and positive-)controls for only three concentrations from each regime. The highest concentration in each case was either that inducing precipitation (for the 4+20-hr treatment, 1200 and 1000 μg/ml in the absence and presence of S9, respectively), or that inducing cytotoxicity (for the 24+0-hr treatment, 500 μg/ml). Frequencies of MNBN cells were not significantly higher than those observed in concurrent vehicle controls for any of the concentrations analysed. As a result, palladium dinitrate can be considered non-clastogenic under the conditions of this study, with and without S9 (Lloyd, 2014b).


Justification for selection of genetic toxicity endpoint
GLP study, conducted according to OECD guidelines.

Justification for classification or non-classification

No evidence of genotoxic activity has been seen in reliable in vitro assays in bacterial or mammalian somatic cells, including GLP guideline studies assessing mutagenic and clastogenic activity. No studies specifically assessing the mutagenic activity in germ cells were identified. However, in support, no effects on reproductive parameters were seen in the combined repeated dose and reproductive/developmental toxicity screening assay on palladium dihydroxide. As such, classification of palladium dinitrate for germ cell mutagenicity is not warranted, according to EU CLP criteria (EC 1272/2008)