tetraammine palladium (II) hydrogen carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a guideline study, to GLP, tetraamminepalladium(II) 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 a guideline study, to GLP, tetraamminepalladium(II) hydrogen carbonate was considered to induce an equivocal clastogenic response to human lymphocytes in vitro, when tested at up to cytotoxic concentrations in the presence (but not absence) of S9 (Wright, 1997).

 

In an OECD guideline study, to GLP, tetraamminepalladium(II) 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 Ames assay, tetraamminepalladium dichloride was not mutagenic in two strains of Salmonella typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of metabolic activation (Suraikina et al., 1979).

In a guideline Ames test, it was concluded that tetraamminepalladium(2+) dichloride did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 64 µg/plate (a toxic concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9) (Ballantyne, 2018).

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).

In limited studies, tetraamminepalladium dichloride (at cytotoxic concentrations) did not induce DNA damage in bacterial SOS chromotests using the E. coli strain PQ37, in the absence of S9 (Gebel et al., 1997 and Lantzsch & Gebel, 1997).

 

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:

9 February-10 April 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study, to GLP. A strain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example Salmonella TA102 or E. coli, was not included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

A strain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example Salmonella TA102 or E. coli, was not included.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

A strain capable of detecting certain oxidising mutagens and/or cross-linking agents, for example Salmonella TA102 or E. coli, 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:

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(II) 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 Salmonella TA102 or E. coli, 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(II) hydrogen carbonate showed no evidence of a dose-related increase in revertant frequency at any dose level in any each strain, either in the presence or absence of S9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Study period:

23 April 2018 - 11 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 100% Tetraamminepalladium(2+) dichloride,hydrate or 97% Tetraamminepalladium(2+) dichloride (based on palladium content of 42.06%)

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S-9 derived from Aroclor 1254-treated male Sprague-Dawley rats

Test concentrations with justification for top dose:

Mutation Experiment 1 (all strains; with and without S9)
5, 16, 50, 160, 500, 1600, 5000 ug/plate

Mutation Experiment 1 (repeat) (all strains; with and without S9)
0.16, 0.5, 1.6, 5, 16, 50, 160 ug/plate

Mutation Experiment 2 (all strains; without S9)
0.26, 0.66, 1.64, 4.1, 10.24, 25.6, 64 ug/plate

Mutation Experiment 2 (all strains; with S9)
0.66, 1.64, 4.1, 10.24, 25.6, 64, 160 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test article stock solutions were prepared by formulating Tetraamminepalladium(2+) dichloride under subdued lighting in purified water with the aid of vortex mixing, to give the maximum required treatment concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm, 0.2 µm pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within approximately 6.5 hours of initial formulation.

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Tetraamminepalladium(2+) dichloride was soluble in water for irrigation (purified water) at concentrations equivalent to at least 50 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Remarks:

2NF for TA98 (-S9); NaN3 for TA100 and TA1535 (-S9); AAC for TA1537 (-S9); MMC for TA102 (-S9); BaP for TA98 (+S9); AAN for TA100, TA1535, TA1537 and TA102 (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation); preincubation (for experiment 2 in the presence of S9).
- 0.1 mL volume additions of test article solution were used for all Experiment 1 treatments, 0.05ml for the positive control treatments

Triplicate plates for test substance, vehicle and positive controls.

Prepared test suspensions were protected from light and used within approximately 6.5 hours of initial formulation.

DURATION
As the results of Experiment 1 were negative, treatments in the presence of S-9 in
Experiment 2 included a pre-incubation step. Quantities of test article, vehicle (reduced to 0.05 mL),
orpositive control, bacteria and S-9 mix detailed above, were mixed
together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C.

Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

DETERMINATION OF CYTOTOXICITY
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity included a marked reduction in revertants compared to the concurrent vehicle controls.

Evaluation criteria:

Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.

The assay was considered to be valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of > (or equal to) 1.5-fold (in strain TA102), > (or equal to) 2-fold (in strains TA98 and TA100) or > (or equal to) 3-fold (in strains TA1535 and TA1537) the concurrent vehicle control, confirming discrimination between different strains, and an active S 9 preparation.

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation
of the plate counts for each treatment were determined. Control counts were
compared with the laboratory’s historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical
analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).
However, adequate interpretation of biological relevance was of critical importance.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp1: evidence of toxicity was observed at 80 µg/plate and above in all strains in the absence of S-9 and in strain TA98 in the presence of S 9, and at 250 µg/plate and above in all other strains in the presence of S-9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Due to the extensive toxicity in Mutation Experiment 1 which resulted in fewer than 5 analysable concentrations for each strain, Mutation Experiment 1 treatments were repeated in all strains, in the absence and presence of S-9, , in order to provide a thorough and robust assessment of mutagenicity of the test article. These treatments were performed using the methodology described above for Mutation Experiment 1, but at lower treatment concentrations. On this occasion, at least 5 analysable concentrations remained for each tester strain.

Data from the initial Experiment 2 treatments of strain TA98 in the absence of S-9 were invalidated due to vehicle control counts that were well above historical control ranges. These strain data are therefore not reported. Strain TA98 treatments were repeated, both in the presence and absence of S-9, in order to provide the Mutation Experiment 2 data which is presented in this report.

From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a single vehicle control replicate plate count in some strains in Mutation Experiment 1 and Mutation Experiment 2. In each case, these vehicle counts were comparable with the other vehicle control replicate plate counts and with the laboratory historical ranges and these data were therefore considered as characteristic and acceptable.

The pH range of all the experimental treatment solution concentrations was within 1 pH on each experimental occasion, apart from the Experiment 2 repeat treatments of strain TA98, where due to an elevated pH value at the lowest treatment concentration, the pH range on this experimental occasion was slightly greater than 1 pH unit. The treatments were considered acceptable based on these pH data.

Conclusions:

In a guideline Ames test, it was concluded that Tetraamminepalladium(2+) dichloride did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 64 µg/plate (a toxic concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Tetraamminepalladium(2+) dichloride was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Tetraamminepalladium(2+) dichloride treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Tetraamminepalladium(2+) dichloride up to 5000 µg/plate. Following these treatments, evidence of toxicity was observed at 50 µg/plate and above in all strains in the absence of S-9, and at 160 µg/plate and above in all strains in the presence of S-9. Due to the extent of this toxicity, fewer than 5 analysable concentrations remained for each strain in each activation condition, and therefore to provide a more thorough and robust assessment of the mutagenicity of Tetraamminepalladium(2+) dichloride in this assay system, Experiment 1 repeat treatments of all the tester strains were performed using treatment concentrations reduced to an estimate of the lower limit of toxicity.

Mutation Experiment 1 repeat treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Tetraamminepalladium(2+) dichloride up to 0.16-160 µg/plate. Following these treatments, evidence of toxicity was again observed at 50 µg/plate and above in all strains in the absence of S-9, and at 160 µg/plate and above in all strains in the presence of S-9. On this occasion, at least 5 analysable concentrations remained for each tester strain.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. A maximum test concentration of 160 µg/plate was used for all strains in the presence of S-9, and 64 µg/plate for all strains in the absence of S-9, these being estimates of the lower limit of toxicity based on the previous experimentation.Narrowed concentration intervals were employed covering the ranges 0.66-160 µg/plate (in the presence of S-9) or 0.26-‑64 µg/plate (in the absence of S-9), in order to examine more closely those concentrations of Tetraamminepalladium(2+) dichloride approaching the toxicity limit. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. Following these treatments, clear evidence of toxicity was observed in all strains treated at the highest concentrations of 64 and 160 µg/plate in the absence and presence of S-9 respectively.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The pH of all concentrations of test article formulation were assessed on each experimental occasion. In each case the range of pH values across the concentration was within or close to 1 pH unit, and were considered acceptable.

Vehicle and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies were consistent with laboratory historical ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Tetraamminepalladium(2+) dichloride treatments of all the test strains in the absence and presence of S-9, there were no notable or concentration-related increases in revertant numbers observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of anyTetraamminepalladium(2+) dichloridemutagenic activity in this assay system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Limited Ames test. Deviations from current OECD recommendations, including requirement for inclusion of additional bacterial tester strains (including strains to detect certain oxidising mutagens and cross-linking agents, i.e. TA102 and/or E.coli), testing with and without mammalian metabolic activation, documentation of actual revertant and toxicity data and actual tested concentrations for follow-up study.

Justification for type of information:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ability to induce mutations in Salmonella typhimurium strains. Limited Ames test.

GLP compliance:

no

Remarks:

Conducted before requirement for GLP.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

without

Test concentrations with justification for top dose:

The test substance was dissolved in distilled water and introduced onto a dish with the test culture in doses of 0.1, 1, 10, 100 or 1000 µg/plate, in the absence of a mammalian metabolic activation system.

Vehicle / solvent:

Distilled water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Remarks:

No details on the identity of the control were given, but it was probably a solvent control.

Evaluation criteria:

For the test substance to be considered mutagenic, a 2.5-fold (or more) increase in the number of colonies must be observed in the plates containing the test substance compared to control .

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The test substance caused less than a 2.5-fold increase in number of colonies in strains TA 98 and TA100 compared with control. No actual revertant data were given.

Conclusions:

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

In a limited Ames assay, tetraamminepalladium dichloride was not mutagenic in two strains of Salmonella typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of metabolic activation.

Executive summary:

In a limited bacterial reverse mutation (Ames) assay, tetraamminepalladium dichloride was not mutagenic in two strains of Salmonella typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of metabolic activation. [Testing in the absence of metabolic activation is not considered critical for inorganics.]

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 April 2018 - 11 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 100% Tetraamminepalladium(2+) dichloride,hydrate or 97% Tetraamminepalladium(2+) dichloride (based on palladium content of 42.06%)

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S-9 derived from Aroclor 1254-treated male Sprague-Dawley rats

Test concentrations with justification for top dose:

Mutation Experiment 1 (all strains; with and without S9)
5, 16, 50, 160, 500, 1600, 5000 ug/plate

Mutation Experiment 1 (repeat) (all strains; with and without S9)
0.16, 0.5, 1.6, 5, 16, 50, 160 ug/plate

Mutation Experiment 2 (all strains; without S9)
0.26, 0.66, 1.64, 4.1, 10.24, 25.6, 64 ug/plate

Mutation Experiment 2 (all strains; with S9)
0.66, 1.64, 4.1, 10.24, 25.6, 64, 160 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test article stock solutions were prepared by formulating Tetraamminepalladium(2+) dichloride under subdued lighting in purified water with the aid of vortex mixing, to give the maximum required treatment concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm, 0.2 µm pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within approximately 6.5 hours of initial formulation.

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Tetraamminepalladium(2+) dichloride was soluble in water for irrigation (purified water) at concentrations equivalent to at least 50 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Remarks:

2NF for TA98 (-S9); NaN3 for TA100 and TA1535 (-S9); AAC for TA1537 (-S9); MMC for TA102 (-S9); BaP for TA98 (+S9); AAN for TA100, TA1535, TA1537 and TA102 (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation); preincubation (for experiment 2 in the presence of S9).
- 0.1 mL volume additions of test article solution were used for all Experiment 1 treatments, 0.05ml for the positive control treatments

Triplicate plates for test substance, vehicle and positive controls.

Prepared test suspensions were protected from light and used within approximately 6.5 hours of initial formulation.

DURATION
As the results of Experiment 1 were negative, treatments in the presence of S-9 in
Experiment 2 included a pre-incubation step. Quantities of test article, vehicle (reduced to 0.05 mL),
orpositive control, bacteria and S-9 mix detailed above, were mixed
together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C.

Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

DETERMINATION OF CYTOTOXICITY
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity included a marked reduction in revertants compared to the concurrent vehicle controls.

Evaluation criteria:

Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.

The assay was considered to be valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of > (or equal to) 1.5-fold (in strain TA102), > (or equal to) 2-fold (in strains TA98 and TA100) or > (or equal to) 3-fold (in strains TA1535 and TA1537) the concurrent vehicle control, confirming discrimination between different strains, and an active S 9 preparation.

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation
of the plate counts for each treatment were determined. Control counts were
compared with the laboratory’s historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical
analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).
However, adequate interpretation of biological relevance was of critical importance.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp1: evidence of toxicity was observed at 80 µg/plate and above in all strains in the absence of S-9 and in strain TA98 in the presence of S 9, and at 250 µg/plate and above in all other strains in the presence of S-9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Due to the extensive toxicity in Mutation Experiment 1 which resulted in fewer than 5 analysable concentrations for each strain, Mutation Experiment 1 treatments were repeated in all strains, in the absence and presence of S-9, , in order to provide a thorough and robust assessment of mutagenicity of the test article. These treatments were performed using the methodology described above for Mutation Experiment 1, but at lower treatment concentrations. On this occasion, at least 5 analysable concentrations remained for each tester strain.

Data from the initial Experiment 2 treatments of strain TA98 in the absence of S-9 were invalidated due to vehicle control counts that were well above historical control ranges. These strain data are therefore not reported. Strain TA98 treatments were repeated, both in the presence and absence of S-9, in order to provide the Mutation Experiment 2 data which is presented in this report.

From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a single vehicle control replicate plate count in some strains in Mutation Experiment 1 and Mutation Experiment 2. In each case, these vehicle counts were comparable with the other vehicle control replicate plate counts and with the laboratory historical ranges and these data were therefore considered as characteristic and acceptable.

The pH range of all the experimental treatment solution concentrations was within 1 pH on each experimental occasion, apart from the Experiment 2 repeat treatments of strain TA98, where due to an elevated pH value at the lowest treatment concentration, the pH range on this experimental occasion was slightly greater than 1 pH unit. The treatments were considered acceptable based on these pH data.

Conclusions:

In a guideline Ames test, it was concluded that Tetraamminepalladium(2+) dichloride did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 64 µg/plate (a toxic concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Tetraamminepalladium(2+) dichloride was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Tetraamminepalladium(2+) dichloride treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Tetraamminepalladium(2+) dichloride up to 5000 µg/plate. Following these treatments, evidence of toxicity was observed at 50 µg/plate and above in all strains in the absence of S-9, and at 160 µg/plate and above in all strains in the presence of S-9. Due to the extent of this toxicity, fewer than 5 analysable concentrations remained for each strain in each activation condition, and therefore to provide a more thorough and robust assessment of the mutagenicity of Tetraamminepalladium(2+) dichloride in this assay system, Experiment 1 repeat treatments of all the tester strains were performed using treatment concentrations reduced to an estimate of the lower limit of toxicity.

Mutation Experiment 1 repeat treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Tetraamminepalladium(2+) dichloride up to 0.16-160 µg/plate. Following these treatments, evidence of toxicity was again observed at 50 µg/plate and above in all strains in the absence of S-9, and at 160 µg/plate and above in all strains in the presence of S-9. On this occasion, at least 5 analysable concentrations remained for each tester strain.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. A maximum test concentration of 160 µg/plate was used for all strains in the presence of S-9, and 64 µg/plate for all strains in the absence of S-9, these being estimates of the lower limit of toxicity based on the previous experimentation.Narrowed concentration intervals were employed covering the ranges 0.66-160 µg/plate (in the presence of S-9) or 0.26-‑64 µg/plate (in the absence of S-9), in order to examine more closely those concentrations of Tetraamminepalladium(2+) dichloride approaching the toxicity limit. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. Following these treatments, clear evidence of toxicity was observed in all strains treated at the highest concentrations of 64 and 160 µg/plate in the absence and presence of S-9 respectively.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The pH of all concentrations of test article formulation were assessed on each experimental occasion. In each case the range of pH values across the concentration was within or close to 1 pH unit, and were considered acceptable.

Vehicle and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies were consistent with laboratory historical ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Tetraamminepalladium(2+) dichloride treatments of all the test strains in the absence and presence of S-9, there were no notable or concentration-related increases in revertant numbers observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of anyTetraamminepalladium(2+) dichloridemutagenic activity in this assay system.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Limited Ames test. Deviations from current OECD recommendations, including requirement for inclusion of additional bacterial tester strains (including strains to detect certain oxidising mutagens and cross-linking agents, i.e. TA102 and/or E.coli), testing with and without mammalian metabolic activation, documentation of actual revertant and toxicity data and actual tested concentrations for follow-up study.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ability to induce mutations in Salmonella typhimurium strains. Limited Ames test.

GLP compliance:

no

Remarks:

Conducted before requirement for GLP.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

without

Test concentrations with justification for top dose:

The test substance was dissolved in distilled water and introduced onto a dish with the test culture in doses of 0.1, 1, 10, 100 or 1000 µg/plate, in the absence of a mammalian metabolic activation system.

Vehicle / solvent:

Distilled water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Remarks:

No details on the identity of the control were given, but it was probably a solvent control.

Evaluation criteria:

For the test substance to be considered mutagenic, a 2.5-fold (or more) increase in the number of colonies must be observed in the plates containing the test substance compared to control .

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The test substance caused less than a 2.5-fold increase in number of colonies in strains TA 98 and TA100 compared with control. No actual revertant data were given.

Conclusions:

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

In a limited Ames assay, tetraamminepalladium dichloride was not mutagenic in two strains of Salmonella typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of metabolic activation.

Executive summary:

In a limited bacterial reverse mutation (Ames) assay, tetraamminepalladium dichloride was not mutagenic in two strains of Salmonella typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of metabolic activation. [Testing in the absence of metabolic activation is not considered critical for inorganics.]

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 1996-21 April 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study, to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-induced S9 prepared in-house from the livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 23.13, 46.25, 92.5, 185, 370, 740, 1480 and 2960 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Media
- Justification for choice of solvent/vehicle: Not applicable

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

at 500 µg/mL for cultures without S9, dissolved in dimethyl sulfoxide

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

at 25 µg/L for cultures with S9, dissolved in culture medium without serum

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Expt 1: 4 hr treatment with harvest following 16 hr expression period
Expt 2: 20 hr continous exposure or 44 hr continuous exposure

Mitosis was stopped by addition of Demecolcine (Colcemid, 0.1 ug/mL)

STAIN (for cytogenetic assays): 5% Gurs Giemsa R66

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per dose, where possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Metaphase spreads were analysed for any increase in chromosome number, gaps, breaks or rearrangements

Statistics:

The frequency of cells with aberrations (including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity response curve with no scorable metaphases at and above 185 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(weak positive) statistically significant increase in frequency of cells with aberrations at 370 µg/mL only (no response at lower doses); did not increase the numbers of polyploid cells significantly

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity response curve (but less steep than without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

slight statistically significant increase in the frequency of cells with gaps at 92.5 µg/mL but not observed in experiment 1 (non-reproducible); no increase in the number of polyploid cells at any dose

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity response curve, 92.5 µg/mL was selected as the maximum scorable dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

statistically significant increase in the frequency of cells with aberrations at 370 µg/mL but not at higher dose of 555 µg/mL; no increase in the number of polyploid cells at any dose

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity response curve, 555 µg/mL was selected as the maximum scorable dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity response curve, 92.5 µg/mL was selected as the maximum scorable dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

statistically significant increase in the frequency of cells with aberrations at 370 µg/mL but not at higher dose of 555 µg/mL; small but significant increase in the numbers of polyploid cells at the 370 µg/mL dose level

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity response curve, 555 µg/mL was selected as the maximum scorable dose level

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: No data

COMPARISON WITH HISTORICAL CONTROL DATA: In lymphocyte control cultures, an acceptable frequency is 0-4% of cells for structural aberrations (including gaps). Excluding gaps, acceptable frequencies are 0-2% for structural aberrations and 0-1% for polyploidy. An observable dose-response relationship is required for modest increases in aberration frequency.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Remarks: Experiment 1: 20 hour harvest (4 hour treatment, 16 hour expression)

Conclusions:

In a guideline study, to GLP, tetraamminepalladium(II) hydrogen carbonate was considered to induce an equivocal clastogenic response to human lymphocytes in vitro, when tested at up to cytotoxic concentrations in the presence (but not absence) of a rat liver metabolic activation (S9) system.

Executive summary:

Tetraamminepalladium(II) hydrogen carbonate was evaluated for clastogenicity in an in vitro chromosome aberration test using human lymphocytes, conducted in accordance with OECD Test Guideline 473, and to GLP.

 

Cells were tested at up to 2960 µg/mL in both the presence and absence of a rat liver metabolic activation (S9) system. Treatment was either continuous (20 or 44 hours), or for 4 hours, followed by a 16-hr expression period.

 

The test material induced statistically significant, but non dose-related, increases in the frequency of cells with chromosome aberrations at one dose level (370 µg/mL) in two separate experiments in the presence of S9. In addition, a small increase in the frequency of numerical aberrations was observed at the same dose level in the extended time point of 44 hours (with S9). No genotoxic response was apparent in the absence of S9.

 

Therefore, tetraamminepalladium(II) hydrogen carbonate was considered to elicit an equivocal clastogenic response to human lymphocytes in vitro in the presense of S9.

Reference 7

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: chromosome damage (micronuclei)

Type of information:

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

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Limited study. Although not a standard (guideline) study, it appears well conducted and scientifically acceptable. However, only a single concetration was analysable (as severe cytotoxicity seen at the highest concentration); whereas current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated.

Justification for type of information:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: method as described by Fenech M (1993) Mut Res 285, 35-44

Principles of method if other than guideline:

Mammalian cytokinesis-block micronucleus assay similar to that described by OECD TG487. Principal difference was that only a single concetration was analysable (as severe cytotoxicity seen at the highest concentration); whereas current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated. Also, test was carried out only in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations of 0, 300 or 600 µM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Blood from healthy, non-smoking donors (aged 25-35-years) was obtained, and the lymphocytes isolated, stained and counted. The lymphocytes were then cultured in medium at a concentration of 500,000/ml, and cell mitosis was stimulated. The test substance was disolved in distilled water and added 24 hr later to the culture in a volume of 20-30 µl. Seventy hours after cell mitosis was stimulated, the cells were harvested, fixed and prepared for microscopy. Micronuclei were scored in 1000 binucleate cells with two nuclei of equal size, and the nuclear division index (NDI) was calculated. Duplicate or triplicate experiments were carried out on different donors.

Evaluation criteria:

Test substance was considered genotoxic if a statistically significant (p<0.05) increase in the mean number of micronuclei were observed.

Statistics:

Number of micronuclei analysed with the X2 test

Species / strain:

mammalian cell line, other: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe cytotoxicity reported at 600 µM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The mean numbers of micronuclei in binucleate cells were 10.7 and 13.0 at concentrations of 0 and 300 µM, respectively, so treatment with tetraamine palladium dichloride produced no statistically significant change from vehicle control. At 600 µM, severe cytotoxicity was seen.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, tetraamminepalladium dichloride did not significantly increase the number of micronuclei in human lymphocytes, in the absence of S9.

Executive summary:

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. [Current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated.]

Reference 8

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: chromosome damage (micronuclei)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Limited study. Although not a standard (guideline) study, it appears well conducted and scientifically acceptable. However, only a single concetration was analysable (as severe cytotoxicity seen at the highest concentration); whereas current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated.

Qualifier:

according to guideline

Guideline:

other: method as described by Fenech M (1993) Mut Res 285, 35-44

Principles of method if other than guideline:

Mammalian cytokinesis-block micronucleus assay similar to that described by OECD TG487. Principal difference was that only a single concetration was analysable (as severe cytotoxicity seen at the highest concentration); whereas current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated. Also, test was carried out only in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations of 0, 300 or 600 µM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Blood from healthy, non-smoking donors (aged 25-35-years) was obtained, and the lymphocytes isolated, stained and counted. The lymphocytes were then cultured in medium at a concentration of 500,000/ml, and cell mitosis was stimulated. The test substance was disolved in distilled water and added 24 hr later to the culture in a volume of 20-30 µl. Seventy hours after cell mitosis was stimulated, the cells were harvested, fixed and prepared for microscopy. Micronuclei were scored in 1000 binucleate cells with two nuclei of equal size, and the nuclear division index (NDI) was calculated. Duplicate or triplicate experiments were carried out on different donors.

Evaluation criteria:

Test substance was considered genotoxic if a statistically significant (p<0.05) increase in the mean number of micronuclei were observed.

Statistics:

Number of micronuclei analysed with the X2 test

Species / strain:

mammalian cell line, other: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe cytotoxicity reported at 600 µM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The mean numbers of micronuclei in binucleate cells were 10.7 and 13.0 at concentrations of 0 and 300 µM, respectively, so treatment with tetraamine palladium dichloride produced no statistically significant change from vehicle control. At 600 µM, severe cytotoxicity was seen.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, tetraamminepalladium dichloride did not significantly increase the number of micronuclei in human lymphocytes, in the absence of S9.

Executive summary:

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. [Current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated.]

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

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:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

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	7	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	0	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	0	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	9	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.

Reference 10

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:

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.

Qualifier:

according to guideline

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	7	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	0	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	0	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	9	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.

Reference 11

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited study. Although not a standard (guideline) study, it appears well conducted and scientifically acceptable

Justification for type of information:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: non standard method as described by Quillardet P and Hofnung M (1985) Mut Res 147, 65-78

Deviations:

yes

Remarks:

As described in Mersch-Sundermann V et al. (1991) Mut Res 252, 51-60; Mersch-Sundermann V et al.(1992) Mut res 278, 1-9; Mersch-Sundermann V et al. (1993) Anticancer Res 13, 2037-2044

Principles of method if other than guideline:

SOS chromotest for DNA damage in the strain E. coli PQ37

GLP compliance:

not specified

Type of assay:

SOS/umu assay

Target gene:

sfiA gene

Species / strain / cell type:

E. coli, other: PQ37

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations range of 10-658 µM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

The SOS chromotest is a short-term assay for the identification of the SfiA gene inducing DNA damage. Test substance was dissolved in distilled water (20 ul) and given to 600 ul portions of the bacterial nutrient broth dilution (5 000 000 CFU/ml). The highest concentration of the substance was taken as "reagent blank" (unless substance was of intensive colour, in which case a blank of each dilution was taken to avoid false negative results). The mixtures were incubated at 37 degrees centigrade for 2.5 hr and afterwards the beta-galactosidase activity (SOS induction) and the alkaline phosphatase activity (toxicity assay) were determined. The maximum induction factor (IF) was the highest IF obtained in the non-cytotoxic range. 4-NQO was used as positive control.

Evaluation criteria:

In order to classify a substance as genotoxic, a continuous increase in beta-galactosidase activity with increasing substance concentration was needed (to avoid false-positive results due to bacteriotoxic effects). Maximum Induction Factor (IFmax) in the non-cytotoxic dose range was also utilised for evaluation as follows:
Not genotoxic: IFmax less than 1.5
Moderately genotoxic: IFmax between 1.5 and 2.0
Genotoxic: IFmax greater than 2.0

Species / strain:

E. coli, other: PQ37

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytoxicity reported at 329 µM

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The maximum induction factor (IFmax) was 1.08. The cytotoxicity threshold was 329 uM.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, tetraamminepalladium dichloride (at cytotoxic concentrations) did not induce DNA damage in a bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9.

Executive summary:

In a limited study, the ability of tetraamminepalladium dichloride (at 10-658 µM) to induce DNA damage in the bacteria Escherichia coli (strain PQ37) was assessed in an SOS chromotest assay, in the absence of a mammalian metabolic (S9) 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

Reference 12

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

supporting study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited study. Although not a standard study, it appears well conducted and scientifically acceptable.

Justification for type of information:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Guideline:

other: non standard method as described by Quillardet P and Hofnung M (1985) Mut Res 147, 65-78

Principles of method if other than guideline:

Bacterial SOS chromotest for DNA damage in the strain Escherichia coli PQ37

GLP compliance:

not specified

Type of assay:

SOS/umu assay

Target gene:

sfiA::lacZ operon gene

Species / strain / cell type:

E. coli, other: PQ37

Metabolic activation:

without

Test concentrations with justification for top dose:

10-658 μM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

The SOS chromotest is a short-term assay for the identification of the SfiA gene inducing DNA damage. Test substance was dissolved in distilled water (20 μl) and given to 600 μl portions of the bacterial nutrient broth dilution (5 000 000 CFU/ml). The highest concentration of the substance was taken as "reagent blank" (unless substance was of intensive colour, in which case a blank of each dilution was taken to avoid false negative results). The mixtures were incubated at 37 degrees centigrade for 2.5 hr and afterwards the beta-galactosidase activity (SOS induction) and the alkaline phosphatase activity (toxicity assay) were determined. The maximum induction factor (IF) was the highest IF obtained in the non-cytotoxic range. 4-NQO was used as positive control.

Evaluation criteria:

In order to classify a substance as genotoxic, a continuous increase in beta-galactosidase activity with increasing substance concentration was needed (to avoid false-positive results due to bacteriotoxic effects). Maximum Induction Factor (IFmax) in the non-cytotoxic dose range was also utilised for evaluation as follows:
Not genotoxic: IFmax less than 1.5
Moderately genotoxic: IFmax between 1.5 and 2.0
Genotoxic: IFmax greater than 2.0

Species / strain:

E. coli, other: PQ37

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity reported at 329 μM

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The maximum induction factor (IFmax) was 1.08. The cytotoxicity threshold concentration was 329 μM.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, tetraamminepalladium dichloride (at cytotoxic concentrations) did not induce DNA damage in a bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9.

Executive summary:

In a limited study, the ability of tetraamminepalladium dichloride (at 10-658 µM) to induce DNA damage in the bacteria Escherichia coli (strain PQ37) was assessed in an SOS chromotest assay, in the absence of a mammalian metabolic (S9) 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.

Reference 13

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited study. Although not a standard (guideline) study, it appears well conducted and scientifically acceptable

Qualifier:

equivalent or similar to guideline

Guideline:

other: non standard method as described by Quillardet P and Hofnung M (1985) Mut Res 147, 65-78

Deviations:

yes

Remarks:

As described in Mersch-Sundermann V et al. (1991) Mut Res 252, 51-60; Mersch-Sundermann V et al.(1992) Mut res 278, 1-9; Mersch-Sundermann V et al. (1993) Anticancer Res 13, 2037-2044

Principles of method if other than guideline:

SOS chromotest for DNA damage in the strain E. coli PQ37

GLP compliance:

not specified

Type of assay:

SOS/umu assay

Target gene:

sfiA gene

Species / strain / cell type:

E. coli, other: PQ37

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations range of 10-658 µM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

The SOS chromotest is a short-term assay for the identification of the SfiA gene inducing DNA damage. Test substance was dissolved in distilled water (20 ul) and given to 600 ul portions of the bacterial nutrient broth dilution (5 000 000 CFU/ml). The highest concentration of the substance was taken as "reagent blank" (unless substance was of intensive colour, in which case a blank of each dilution was taken to avoid false negative results). The mixtures were incubated at 37 degrees centigrade for 2.5 hr and afterwards the beta-galactosidase activity (SOS induction) and the alkaline phosphatase activity (toxicity assay) were determined. The maximum induction factor (IF) was the highest IF obtained in the non-cytotoxic range. 4-NQO was used as positive control.

Evaluation criteria:

In order to classify a substance as genotoxic, a continuous increase in beta-galactosidase activity with increasing substance concentration was needed (to avoid false-positive results due to bacteriotoxic effects). Maximum Induction Factor (IFmax) in the non-cytotoxic dose range was also utilised for evaluation as follows:
Not genotoxic: IFmax less than 1.5
Moderately genotoxic: IFmax between 1.5 and 2.0
Genotoxic: IFmax greater than 2.0

Species / strain:

E. coli, other: PQ37

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytoxicity reported at 329 µM

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The maximum induction factor (IFmax) was 1.08. The cytotoxicity threshold was 329 uM.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, tetraamminepalladium dichloride (at cytotoxic concentrations) did not induce DNA damage in a bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9.

Executive summary:

In a limited study, the ability of tetraamminepalladium dichloride (at 10-658 µM) to induce DNA damage in the bacteria Escherichia coli (strain PQ37) was assessed in an SOS chromotest assay, in the absence of a mammalian metabolic (S9) 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

Reference 14

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited study. Although not a standard study, it appears well conducted and scientifically acceptable.

Qualifier:

no guideline available

Guideline:

other: non standard method as described by Quillardet P and Hofnung M (1985) Mut Res 147, 65-78

Principles of method if other than guideline:

Bacterial SOS chromotest for DNA damage in the strain Escherichia coli PQ37

GLP compliance:

not specified

Type of assay:

SOS/umu assay

Target gene:

sfiA::lacZ operon gene

Species / strain / cell type:

E. coli, other: PQ37

Metabolic activation:

without

Test concentrations with justification for top dose:

10-658 μM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

The SOS chromotest is a short-term assay for the identification of the SfiA gene inducing DNA damage. Test substance was dissolved in distilled water (20 μl) and given to 600 μl portions of the bacterial nutrient broth dilution (5 000 000 CFU/ml). The highest concentration of the substance was taken as "reagent blank" (unless substance was of intensive colour, in which case a blank of each dilution was taken to avoid false negative results). The mixtures were incubated at 37 degrees centigrade for 2.5 hr and afterwards the beta-galactosidase activity (SOS induction) and the alkaline phosphatase activity (toxicity assay) were determined. The maximum induction factor (IF) was the highest IF obtained in the non-cytotoxic range. 4-NQO was used as positive control.

Evaluation criteria:

In order to classify a substance as genotoxic, a continuous increase in beta-galactosidase activity with increasing substance concentration was needed (to avoid false-positive results due to bacteriotoxic effects). Maximum Induction Factor (IFmax) in the non-cytotoxic dose range was also utilised for evaluation as follows:
Not genotoxic: IFmax less than 1.5
Moderately genotoxic: IFmax between 1.5 and 2.0
Genotoxic: IFmax greater than 2.0

Species / strain:

E. coli, other: PQ37

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity reported at 329 μM

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The maximum induction factor (IFmax) was 1.08. The cytotoxicity threshold concentration was 329 μM.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, tetraamminepalladium dichloride (at cytotoxic concentrations) did not induce DNA damage in a bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9.

Executive summary:

In a limited study, the ability of tetraamminepalladium dichloride (at 10-658 µM) to induce DNA damage in the bacteria Escherichia coli (strain PQ37) was assessed in an SOS chromotest assay, in the absence of a mammalian metabolic (S9) 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.

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 and GLP, tetraamminepalladium(II) 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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study performed between 9 February and 12 March 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD and EU guideline study, to GLP. No details on test material purity and stability. Although no bone marow cytotoxicity observed, premature deaths and clincial signs indicate that systemic absorption had ocurred.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DG0475
- Expiration date of the lot/batch: no data
- Purity: no data. Responsibility of sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: no data. Responsibility of sponsor

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: freshly prepared as required as a suspension in arachis oil

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Albino Crl:CD-1 (1CR)BR strain mice

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, Margate, Kent, UK.
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 24-30 g (group means 26.0-27.7 g)
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: up to 7 animals in solid-floor polypropylene cages with woodflakes bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 50-52
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 12.5, 25 or 50 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg

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: based on MTD from range-finding study

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:

In an in vivo study, conducted to OECD guideline and GLP, tetraamminepalladium(II) 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(II) hydrogen carbonate to produce damage to chromosomes or aneuploidy when administered orally (by gavage) to mice. The study design complied with OECD Test Guideline 474 and EU Method B12, and was to GLP.

 

Following a range-finding study, groups of seven male mice were administered 125, 250 or 500 (the MTD) mg/kg bw of the test material via gavage 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(II) 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

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

Tetraamminepalladium(II) 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). 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(II) hydrogen carbonate showed no evidence of a dose-related increase in revertant frequency at any dose level 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 Salmonella TA102 or E. coli. 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). In a guideline Ames test, it was concluded that tetraamminepalladium(2+) dichloride did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 64 µg/plate (a toxic concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9) (Ballantyne, 2018).

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).

Tetraamminepalladium(II) 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(II) 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).

 

Tetraamminepalladium(II) hydrogen carbonate was evaluated for clastogenicity in an in vitro chromosome aberration test using human lymphocytes, conducted in accordance with OECD Test Guideline 473, and to GLP. Cells were tested at up to 2960 µg/mL in both the presence and absence of a rat liver metabolic activation (S9) system. Treatment was either continuous (20 or 44 hours), or for 4 hours, followed by a 16-hr expression period. The test material induced statistically significant, but non dose-related, increases in the frequency of cells with chromosome aberrations at one dose level (370 µg/mL) in two separate experiments in the presence of S9. In addition, a small increase in the frequency of numerical aberrations was observed at the same dose level in the extended time point of 44 hours (with S9). No genotoxic response was apparent in the absence of S9. Therefore, tetraamminepalladium(II) hydrogen carbonate was considered to elicit an equivocal clastogenic response to human lymphocytes in vitro in the presence of S9 (Wright, 1997).

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(II) hydrogen carbonate to produce damage to chromosomes or aneuploidy when administered orally (by gavage) to mice. The study design complied with OECD Test Guideline 474 and EU Method B12, and was to GLP. Following a range-finding study, groups of seven male mice were administered 125, 250 or 500 (the MTD) mg/kg bw of the test material via gavage 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(II) 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(II)dichloride 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 classification or non-classification

Based on the existing data set, tetraamminepalladium(II) hydrogen carbonate does not meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008).