Tetraamminepalladium(2+) dinitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a guideline study, to GLP, tetraamminepalladium hydrogen carbonate was not mutagenic in a bacterial reverse mutation (Ames) assay using five Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537 and TA1538), when tested at up to cytotoxic concentrations in the presence and absence of a rat liver metabolic activation (S9) system (Thompson, 1997).

 

Link to relevant study records

Reference

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:

9 February-10 April 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP, on closely-related surrogate. Astrain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example TA102, was not included.

Justification for type of information:

Tetraamminepalladium hydrogen carbonate is considered to fall within the scope of the read-across category "tetraamminepalladium salts". See IUCLID section 13 for full read-across justification report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Astrain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example TA102, was not included.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Astrain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example TA102, was not included.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolising system (S9)

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 µg/plate in preliminary cytotoxicity test
0, 0.15 (Expt 1 only), 0.5, 1.5, 5, 15, 50 (-S9)
0, 1.5, 5, 15, 50, 150, 500 (Expt 1 only) (+S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Test guideline recommends use of aqueous solvent wherever possible

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA 1537 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA 98 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenyl enediamine

Remarks:

5 µg/plate for TA 1538 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 and 5 µg/plate for TA 100 and 1535 respectively (both without S9)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

1, 2, 0.5, 0.5 and 2 µg/plate for TA 100, 1535, 1538, 98 and 1537 strains respectively (all with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Not applicable
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable
- 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: 3 (Experiment carried out twice)

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in no. of revertant colonies/thining of background lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable

Evaluation criteria:

Positive results should have a dose-related and statistically significant increase in mutation rate in one or more bacterial strains with or without S9. To be considered negative, the number of induced revertants should be less than twofold compared to spontaneous revertants (controls).

Statistics:

No data

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial lawn at 50 and 150 µg/plate and above for tester strains without and with metabolic activation respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial lawn at 50 and 150 µg/plate and above for tester strains without and with metabolic activation respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial lawn at 50 and 150 µg/plate and above for tester strains without and with metabolic activation respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial lawn at 50 and 150 µg/plate and above for tester strains without and with metabolic activation respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial lawn at 50 and 150 µg/plate and above for tester strains without and with metabolic activation respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The dose range used was 0, 50, 150, 500, 1500 and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial lawn at 50 and 150 µg/plate and above for tester strains without and with metabolic activation respectively

Remarks on result:

other: strain/cell type: TA 100

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In a guideline study, to GLP, tetraamminepalladium hydrogen carbonate was not mutagenic in a bacterial reverse mutation (Ames) assay using five Salmonella typhimurium strains (TA 98, TA100, TA1535, TA1537 and TA1538), when tested at up to cytotoxic concentrations in the presence and absence of a rat liver metabolic activation (S9) system.

Executive summary:

Tetraamminepalladium hydrogen carbonate was assessed for potential mutagenic activity in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471, and to GLP. The test compound was tested in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538). (However, a strain capable of detecting cross-linking mutagens, for example TA102, was not included.)

 

The dose ranges were determined in a preliminary assay for cytotoxicity and were 0.15-50 and 1.5-500 µg/plate with and without the addition of a rat liver homogenate metabolising (S9) system. All assays were carried out in triplicate, at up to 50 and 500 µg/plate in the absence and presence of S9, respectively. The experiment was repeated.

 

Tetraamminepalladium hydrogen carbonate showed no evidence of a dose-related increase in revertant frequency at any dose levels in any each strain, either in the presence or absence of S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies conducted in humans were identified (although in vitro studies using human lymphocytes are described below).

 

Tetraamminepalladium hydrogen carbonate was assessed for potential mutagenic activity in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471, and to GLP. The test compound was tested in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538). (However, a strain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example TA102, was not included.) The dose ranges were determined in a preliminary assay for cytotoxicity and were 0.15-50 and 1.5-500 µg/plate with and without the addition of a rat liver homogenate metabolising (S9) system. All assays were carried out in triplicate, at up to 50 and 500 µg/plate in the absence and presence of S9, respectively. The experiment was repeated. Tetraamminepalladium hydrogen carbonate showed no evidence of a dose-related increase in revertant frequency at any dose levels in any strain, either in the presence or absence of S9 (Thompson, 1997). [This study lacks a bacterial strain susceptible to oxidative mutagenesis or cross-linking agents, for example TA102. However, tetraamminepalladium compounds are not expected to cause oxidative damage or to be cross-linking agents. This is based primarily on the negative Ames results in studies utilising TA102/E.coli, and negative SOS Chromotest with E.coli, conducted with related palladium compounds and also the reassuring results (mutagenicity and cytogenicity) in the available in vitro studies (including SOS Chromotest and in mammalian cells) as well as an in vivo study in mice with various tetraamminepalladium compounds (see below for details). As such this Ames study is considered sufficient for satisfying this REACH requirement and as support for the non-classification of tetraamminepalladium compounds for mutagenicity].

 

In support, in a limited Ames test, tetraamminepalladium dichloride was not mutagenic in two strains of S. typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of S9 (Suraikina et al., 1979). [Testing in the absence of metabolic activation is not considered critical for inorganics.]

 

Tetraamminepalladium diacetate was assessed for its ability to induce mutations at the hprt locus in an in vitro mouse lymphoma assay conducted in accordance with OECD Test Guideline 476 and to GLP. L5178Y mouse lymphoma cells were exposed to test material for 3 hr in two independent experiments, each in the absence and presence of S9. Concentrations of 75 to 500 μg/mL and 50 to 450 μg/mL (Experiment 1, without and with S9, respectively) and 25 to 400 μg/mL and 50 to 500 μg/mL (Experiment 2, without and with S9, respectively) were used. Cytotoxicity was observed seven days after treatment at the highest tested levels, with concentrations of 350-400 μg/mL (Experiment 1, without S9); 450 μg/mL (Experiment 1, with S9); 400 μg/mL (Experiment 2, without S9) and 500 μg/mL (Experiment 2, with S9) being considered too toxic for selection to determine viability and 6TG resistance. Statistically significant increases in mutant frequency (MF) over the concurrent vehicle control value were observed in Experiment 2 in the presence of S9, at the highest two concentrations analysed (380 and 450 μg/mL) but not in the absence of S9 or with or without S9 in Experiment 1. The mean MF values at 380 and 450 μg/mL were 3.84 and 2.34 mutants/10^6 viable cells, respectively, compared to the concurrent vehicle control MF value of 1.11. These increases were small in magnitude but statistically significant as they were compared to a low vehicle control value (the historical control value was 3.25). Furthermore, there was no evidence of reproducibility between experiments in the presence of S9 and no statistically significant linear trends in Experiments 1 and 2, therefore the small, non-reproducible increases seen in Experiment 2 were considered not biologically relevant. Overall tetraamminepalladium diacetate solution did not induce mutation at the hprt locus of mouse lymphoma (L5178Y) cells when tested up to toxic concentrations in two independent experiments, each in the absence and presence of S9 (Lloyd, 2015).

In a limited study, the ability of tetraamminepalladium dichloride to induce micronuclei in human peripheral mononuclear blood cells (lymphocytes) was assessed, in the absence of added metabolic (S9) activation. The mean numbers of micronuclei in binucleate cells were 10.7 and 13.0 at concentrations of 0 and 300 µM, respectively. As such, treatment produced no statistically significant change from the vehicle (distilled water) control. At 600 µM, severe cytotoxicity was seen and no assessment of chromosome damage was possible. In conclusion, tetraamminepalladium dichloride did not induce chromosome damage in a limited cytokinesis-block micronucleus test with human lymphocytes that employed only one single viable test concentration (Gebel et al., 1997). [Current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated.]

 

In a limited study, the ability of tetraamminepalladium dichloride (at 10-658 µM) to induce DNA damage in the bacterium Escherichia coli (strain PQ37) was assessed in an SOS chromotest assay, in the absence of any mammalian metabolic activation system. Cytotoxicity was seen at 329 µM. A maximum induction factor (IFmax, in the absence of cytotoxicity) of 1.08 was reported, indicating that the test substance had no genotoxic effect. In conclusion, the test substance did not show any ability to induce DNA damage in a bacterial SOS chromotest in E. coli PQ37, without S9 (Gebel et al., 1997; Lantzsch and Gebel, 1997).

  

An in vivo study was performed to assess the potential of tetraamminepalladium hydrogen carbonate to produce damage to chromosomes or aneuploidy when administered orally to mice. The study design complied with OECD Test Guideline 474 and EU Method B12. Following a range-finding study, groups of seven male mice were given 125, 250 or 500 (the MTD) mg/kg bw of the test material and killed 24 or 48 hours later for analysis of micronuclei in polychromatic and normochromatic erythrocytes. Further groups of mice were given arachis oil or cyclophosphamide as vehicle and positive controls, respectively. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24- or 48-hr test material dose groups when compared to their concurrent control groups. However, the presence of premature deaths and clinical signs indicated that systemic absorption had occurred. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes, confirming the sensitivity of the test system. In conclusion, tetraamminepalladium hydrogen carbonate failed to produce evidence of chromosome damage following oral gavage at up to 500 mg/kg bw, under the conditions of the test (Durward, 1998).

Tetraamminepalladium hydrogen carbonate and diacetate are considered to fall within the scope of the read-across category "tetraamminepalladium salts". See IUCLID section 13 for full read-across justification report.

 

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, including GLP guideline studies assessing mutagenic and clastogenic activity, nor in a reliable in vivo study assessing chromosome damage. No studies specifically assessing the mutagenic activity in germ cells were identified. However, no effects on reproductive parameters were seen in the reproductive/developmental toxicity screening assay. As such, classification of tetraamminepalladium dichloride for germ cell mutagenicity is not warranted, according to EU CLP criteria (EC 1272/2008).

Via read-across, the same conclusion applies to tetraamminepalladium dinitrate (cfr IUCLID Section 13).