Registration Dossier

Administrative data

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).

 

In an OECD guideline study, to GLP, tetraamminepalladium diacetate solution failed to induce mutations 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, tetraamminepalladium dichloride did not significantly increase the incidence of micronuclei in human lymphocytes, in the absence of S9 (Gebel et al., 1997).

 

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Remarks:
Read-across from study conducted on a member of the "tetraamminepalladium salts" category, tetraamminepalladium hydrogen carbonate
Adequacy of study:
weight of evidence
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:
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:
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. certificate)
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:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Remarks:
Read-across from study conducted on a member of the "tetraamminepalladium salts" category, tetraamminepalladium diacetate
Adequacy of study:
key study
Study period:
16 September - 3 December 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, to GLP, with minor deviations that are not considered to affect the validity of the study.
Justification for type of information:
Tetraamminepalladium diacetate 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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Prior to plating Experiment 2 in the presence of S9, some of the cell suspension for vehicle control replicate ‘A’ was lost due to spillage, therefore three (not four) mutation plates were plated.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y tk+/- (3.7.2C) mouse lymphoma cells sourced from Burroughs Wellcome Co. Stocks were stored in liquid nitrogen prior to use. Each batch of frozen cells was purged of mutants and confrimed to be mycoplasma free
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver post-mitochondrial fraction (S-9) from male Sprague-Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Cytotoxicity range-finder experiment: 91.47, 182.9, 365.9, 731.8, 1464 and 2927 μg/mL (both with and without S9)
Experiment 1: 75, 150, 200, 240, 270, 300, 325, 350, 375, 400 and 500 μg/mL (without S9) and 50, 100, 150, 200, 240, 270, 300, 325, 350, 375 and 450 μg/mL (with S9)
Experiment 2: 25, 50, 100, 150, 200, 230, 260, 290, 320, 350 and 400 μg/mL (without S9) and 50, 100, 150, 200, 230, 260, 290, 320, 350, 380, 450 and 500 μg/mL (with S9)
Vehicle / solvent:
Purified water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline 1-oxide (NQO; without S9) and benzo[a]pyrene (B[a]P; with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: At least 10^7 cells in a volume of 17.0 mL of RPMI 1640 with 5% heat-inactivated horse serum were placed in a series of sterile disposable 50 mL centrifuge tubes. For each treatment 2.0 mL vehicle or test article or 0.2 mL positive control solution plus 1.8 mL purified water was added. S9 mix or 150 mM KCl was added as appropriate. Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL. After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 x g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 x g) for 5 minutes and resuspended in 20 mL RPMI 1640 with 10% serum. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.

DURATION
- Preincubation period: Until the cells were growing well
- Exposure duration: 3 hr for all experiments
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12-13 days
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): 6-thioguanine
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: The experiment was performed in duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test substance was measured by calculating the relative survival percentages. Wells containing viable clones were identified by eye using background illumination and counted.
Evaluation criteria:
The assay was considered valid if both the mutant frequency (MF) in the vehicle control cultures fell within the normal range (up to three times the historical control value) and at least one concentration of each of the positive control chemicals induced a clear, unequivocal increase in MF.
For valid data, the test article was considered to induce forward mutations at the hprt locus if: a) the MF at one or more of the concentrations was significantly greater than that of the vehicle control (p<=0.05); b) there was a significant concentration-relationship as indicated by the linear trend analysis (p<=0.05); c) the effects were reproducible.
Statistics:
Dunnett's test (one-sided) was used for the analysis of the statitical significance of increased mutant frequencies at different concentrations relative to controls.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Seven days after treatment, concentrations of 350-400 μg/mL (Exp 1, without S9); 450 μg/mL (Exp 1, with S9); 400 μg/mL (Exp 2, without S9) and 500 μg/mL (Exp 2, with S9) were considered too toxic for selection to determine viability and 6TG resistance.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No marked changes in osmolality or pH were observed at the highest concentration tested in either the range-finding test or main experiment (see table 1).

In a cytotoxicity range-finder experiment, the highest concentrations to give a relative survival percentage of at least 10% were 182.9 μg/mL (without S9) and 365.9 μg/mL (with S9) (see table 2).

In Experiment 1, precipitation occurred following the 3-hr treatment period in the highest two concentrations tested in the absence of S9 (400 and 500 μg/mL) and the highest of these was discarded. Seven days after treatment, the highest three remaining concentrations in the absence of S9 (350 to 400 μg/mL) and the highest concentration in the presence of S9 (450 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance (see table 3).

In Experiment 2, seven days after treatment, the highest concentrations tested in the absence and presence of S9 (400 and 500 μg/mL, respectively) were considered too toxic for selection to determine viability and 6TG resistance (see table 4).

When tested up to toxic concentrations for 3 hr in the absence and presence of S9 in Experiment 1 and in the absence of S9 in Experiment 2, no statistically significant increases in MF were observed at any concentration analysed. A statistically significant linear trend (p<=0.01) was observed in the absence of S9 in Experiment 2, but as there were no statistically significant increases in MF at any concentration analysed in this experiment this observation was considered not biologically relevant.

In Experiment 2 in the presence of S9, statistically significant increases in MF over the concurrent vehicle control value were observed at the highest two concentrations analysed (380 and 450 μg/mL, giving 29% and 9% relative survival (RS), respectively) but there was no statistically significant linear trend. 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. At the time of Experiment 2, the historical mean vehicle control MF value was 3.25, therefore any vehicle control value below 9.75 (3.25 x 3) would be considered acceptable. The MF values at 380 and 450 μg/mL were very close to (or below) 3.25, but were compared against a low vehicle control MF, therefore the increases over the vehicle control MF were significant despite being small in magnitude. 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: pH measurements of stock formulations

 Experiment  Concentration of stock formulation (mg/mL)  pH of stock formulation
Range-finder   37.10  7.94
 1  5.00  8.02
 2  5.00  8.11

Table 2: Cytotoxicity data from range-finding test

 Concentration (μg/mL)  Relative survival (%) [without S9]  Relative survival (%) [with S9]
 0 100 100 
 91.47 87  68 
 182.9 52  36 
 365.9 20 *
 731.8  0 *  **

* Precipitation observed at the end of the treatment incubation period

** Not plated due to precipitation

Table 3: Cytotoxic and mutagenic response in experiment 1

  Concentration (μg/mL)  3-hr treatment [without S9]      Concentration (μg/mL)  3-hr treatment [with S9]   
   RS (%)  MF (*)    RS (%)  MF (*)
 0 100  4.10  100  2.29 
 75 76  1.48  50  107  2.78 
 150 49  3.74  100  81  1.49 
 200 33  2.91  150  68  0.79 
 240 20  3.40  200  48  3.71 
 270 19  4.23  240  43  2.16 
 300 17  5.88  270  36  3.41 
 325 10  2.25  300  28  1.59 
 NQO 0.15 31  43.3  325  25  3.21 
 NQO 0.20  26 54.47  350  21  2.69 
       375 13  3.13 
       B[a]P 2 79  9.39 
       B[a]P 3 42  42.47 

RS: relative survival

MF: mutant frequency

* Mutants per 10^6 viable cells 7 days after treatment

Table 4: Cytotoxic and mutagenic response in experiment 2

  Concentration (μg/mL)  3-hr treatment [without S9]      Concentration (μg/mL)  3-hr treatment [with S9]   
   RS (%)  MF (*)    RS (%)  MF (*)
0 100 0.84  100  1.11 
 25 75  0.74  50  90  1.75 
 50 78  1.19  100  90  2.45 
 100 77  1.11  150  74  1.72 
 150 48  1.18  200  67  2.40 
 200 40  1.09  230  57  0.92 
 230 38  1.70  260  51  1.22 
 260 32  1.20  290  42  0.93 
 290 26  1.90  320  33  1.55 
 320 16  3.63  350  24  1.85 
 350 12  2.15  380  29  3.84 
 NQO 0.15 30  25.39  450  2.34 
 NQO 0.20  20 34.30  B[a]P 2  92  14.93 
      B[a]P 3 63  36.20 

* Mutants per 10^6 viable cells 7 days after treatment

Conclusions:
Interpretation of results (migrated information):
negative

In an OECD guideline study, to GLP, tetraamminepalladiumdiacetate solution failed to induce mutations at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, each in the absence and presence of S9.
Executive summary:

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.

Mouse lymphoma (L5178Y) 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.

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

Genetic toxicity in vivo

Description of key information

In an in vivo study, conducted to comply with OECD Test Guideline 474, tetraamminepalladium hydrogen carbonate failed to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice following oral gavage at up to 500 mg/kg bw (Durward, 1998).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Remarks:
Read-across from study conducted on a member of the "tetraamminepalladium salts" category, tetraamminepalladium hydrogen carbonate
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD and EU guideline study. Unfortunately, a number of pages are missing from the study report, therefore it has not been possible to verify any deviations.
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: group means 26.0-27.7 g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data
Duration of treatment / exposure:
Animals were given a single oral dose
Frequency of treatment:
Single dose
Post exposure period:
Animals were killed 24 and 48 hours following treatment
Remarks:
Doses / Concentrations:
125, 250, 500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Groups of seven male mice per treatment group; Seven males recieved the vehicle (arachis oil) control; Five males recieved the positive (cyclophosphamide) control.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
Bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes scored for the presence of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: maximum tolerated dose (MTD) was 500 mg/kg bw, in the dose-ranging finding study. Doses of 125 and 250 mg/kg bw were considered appropriate as the two lower dose levels.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were killed 24 or 48 hours later.

DETAILS OF SLIDE PREPARATION: bone marrow extracted and smear preparations made and stained.

METHOD OF ANALYSIS: Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei

OTHER:
Evaluation criteria:
A statistically significant increase in the frequency of micronucleated PCEs compared to the concurrent vehicle control group was considered evidence of a positive effect.
Statistics:
PCE/NCE ratio
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Premature deaths were seen in the 24-hour 500 mg/kg bw (2) and 125 mg/kg bw (1) test material groups. Clinical signs were observed in animals dosed with the test material at and above 125 mg/kg bw in both the 24 and 48-hour groups, where applicable.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hr test material groups when compared to their concurrent vehicle control groups. However, the presence of premature deaths and clinical observations indicated that systemic absorption had occurred.

There were premature deaths seen in the 24-hr 500 mg/kg bw (two animals) and 125 mg/kg bw (one animal) test material dose groups. Clinical signs were observed in animals dosed with the test material at and above 125 mg/kg bw in both the 24- and 48-hr groups, where applicable. Clinical signs included: hunched posture, ptosis, pilo-erection, lethargy, pallor of the extremities, splayed gait, tiptoe gait, decreased respiratory rate, laboured respiration, ataxia, noisy respiration, gasping respiration, increased lacrimation and increased salivation. It was considered that the loss of animals due to premature death did not effect the integrity of the study, with at least five analysable animals being available per group as recommended in the OECD guidelines.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Micronucleus study - summary of group mean data:

 TREATMENT GROUP     Number of PCE with micronuclei per 2000 PCE     PCE/NCE ratio
   Group mean  SD  Group mean  SD

Vehicle control

48-hr sampling time

 1.4  1.7

 1.47

 0.55

Vehicle control

24-hr sampling time

 1.1  0.7

 1.38

 0.53

Positive control

24-hr sampling time 

 25.0***  4.6

 1.47

 0.26

Tetraamminepalladium hydrogen carbonate

500 mg/kg bw

48-hr sampling time 

 0.9  0.7

 0.98

 0.58

Tetraamminepalladium hydrogen carbonate(a)

500 mg/kg bw

24-hr sampling time

1.8 

 1.9

 1.67

 1.16

Tetraamminepalladium hydrogen carbonate

500 mg/kg bw

24-hr sampling time

 1.7

 1.5

 1.13

 0.16

Tetraamminepalladium hydrogen carbonate(b)

500 mg/kg bw

24-hr sampling time

 0.5

 0.5

 1.15

 0.58

Key:

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

*** = p<0.001

a = data from five animals

b = data from six animals

Conclusions:
Interpretation of results (migrated information): negative
In an in vivo guideline study, tetraamminepalladium hydrogen carbonate failed to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice following oral gavage at up to 500 mg/kg bw.
Executive summary:

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.

Tetraamminepalladium hydrogen carbonate is closely-related to tetraamminepalladium dichloride, and is considered a suitable surrogate for read-across for this endpoint. The proposed read-across is appropriate because it is expected that the target and source substances undergo biotransformation to a common product. In solution, the hydrogen carbonate and chloride anions are expected to dissociate from the tetraamminepalladium cation; thus, this can be regarded as the common product and toxicologically-active species of both salts. The chloride and hydrogen carbonate counterions would not have an impact on the overall clastogenicity of the target or source substance, respectively. Therefore, it is considered that use of in vivo micronuclei induction data obtained on a test of tetraamminepalladium hydrogen carbonate to fill a gap in the standard information requirements for tetraamminepalladium dichloride is scientifically justified and suitably reliable.

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

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 in bacterial or somatic cells, 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).