Diamminedichloropalladium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a OECD Test Guideline 471 study, to GLP, diamminedichloropalladium was not mutagenic in a bacterial reverse mutation (Ames) assay using five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537), when tested at up to cytotoxic concentrations in the presence and absence of a rat liver metabolic activation (S9) system (McGarry, 2012a).

In an OECD Test Guideline 476 study, to GLP, diamminedichloropalladium failed to induce biologically relevant increases in mutations at the hprt locus of mouse lymphoma (L5178Y) cells when tested up to the limits of solubility in two independent experiments, each in the absence and presence of S9 (Lloyd, 2013).

In an in vitro mammalian cell micronucleus test, conducted according to GLP and OECD Test Guideline 487, diamminedichloropalladium (at up to 40 µg/mL; limited by solubility) did not cause a treatment-related increase in the frequency of micronuclei in cultured human peripheral blood lymphocytes, with or without S9 (Watters, 2012).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January 2012 (initiation) - 12 March 2012 (study completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study, to GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Very minor deviations documented; would not affect study integrity or results.

Qualifier:

according to guideline

Guideline:

other: UKEMS (Gatehouse et al. 1990, see below for full reference)

Deviations:

yes

Remarks:

Very minor deviations documented; would not affect study integrity or results.

Qualifier:

according to guideline

Guideline:

other: ICH 2A (1995) and ICH 2B (1997)

Deviations:

yes

Remarks:

Very minor deviations documented; would not affect study integrity or results.

Principles of method if other than guideline:

Guideline methods:
Gatehouse D G, Wilcox P, Forster R, Rowland I R and Callander R D (1990) Bacterial mutation assays. In "Basic Mutagenicity Tests UKEMS Recommended Procedures". Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part I Revised. Ed D J Kirkland. Cambridge University Press, pp 13-61.

ICH-S2A (1995). “Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals”.

ICH-S2B (1997). “Standard Battery for Genotoxicity Tests for Pharmaceuticals”.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

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:

S9

Test concentrations with justification for top dose:

EXPERIMENT 1:
0, 0.126 (in the absence of S9 only), 0.4, 1.26, 4, 12.6, 40, 126 and 400 (in the presence of S9 only) ug/plate - in the presence and absence of S9 unless stated otherwise.

EXPERIMENT 2:
1.29, 3.226, 8.064, 20.16, 50.4 and 126 ug/plate - in the absence of S9
OR
4.096, 10.24, 25.6, 64, 160 and 400 ug/plate - in the presence of S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (dimethylformamide)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Diamminedichloropalladium was soluble in dimethylformamide (DMF) at concentrations of at approximately 4.00 mg/mL, the highest achievable concentration of the series tested. The other vehicles tested were purified water, acetone, ethanol, RPMI 1640 culture medium or tetrahydrofuran. Diamminedichloropalladium solubility in dimethyl sulphoxide (DMSO) was not performed at the request of the sponsor as certain chlorinated platinum analogues have been shown to react with DMSO in a manner which was considered likely to impact the scientific integrity of genotoxicity studies.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-NF; NaN3; AAC; MMC; B[a]P; AAN

Details on test system and experimental conditions:

For all assays, bacteria were cultured at 37±1 degrees C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.

The platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1 degrees C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1 degrees C in the dark for 3 days.

As the results of the first experiment were negative, treatments in the presence of S 9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution, bacteria and S 9 mix detailed above, plus an additional 0.5 mL of 500 mM sodium phosphate buffer (pH 7.4) were mixed together and incubated for 20 minutes at 37±1 degrees C, with shaking, before the addition of 2 mL of 1.125% molten agar at 46±1 degrees C. For the pre-incubation treatments, positive control volumes were reduced to 0.05 mL. Plating of these treatments then proceeded as for the normal plate incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay. (The addition of 0.5 mL of 500 mM sodium phosphate buffer (pH 7.4) to these Experiment 2 treatments was employed to reduce the solvent concentration during the pre-incubation period. DMF, and some other organic solvents, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By employing the modification indicated, the DMF concentration in the pre-incubation mix was decreased, and it was hoped that this would minimise or eliminate any toxic effects of the solvent that may have otherwise occurred. In order to 'correct' for the additional volume in the pre incubation mix, these were plated out using 2 mL of 1.125% soft agar, therefore the additions to each plate were comparable to that of the plate-incorporation treatments. )

Following incubation, the plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted either electronically using a Sorcerer Colony Counter (Perceptive Instruments), or manually where confounding factors such as a damaged plate or bubbles in the agar affected the accuracy of the automated counter.

Test concentrations and positive controls were tested in triplicate; negative controls were tested in quintuplicate.

The pH of the stock formulation prior to treatment was 12.03 in Experiment 1 and 12.71 in Experiment 2.

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

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test (which compared the counts at each concentration with the control), an increase in revertant numbers gave a statistically significant response (p less than or equal to 0.01) which was concentration related up to limiting levels (toxicity), as checked by non-statistical analysis.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Analysed at the 1% level using Dunnett's test (for each test concentration compared to control).

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment 1, complete toxicity was seen in all strains from 40 ug/plate in the absence of S9 and from 126 ug/plate in its presence. In experiment 2, complete toxicity was seen in all strains from 50.4 ug/plate in the absence of S9 and from 160 ug/plate in its presence

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Diamminedichloropalladium was not mutagenic in an Ames assay conducted according to OECD (and other relevant) guidelines and using five strains of Salmonella typhimurium, when tested up to limits of toxicity in the presence and absence of an S9 metabolic activation fraction (400 ug/plate in its presence and 126 ug/plate in its absence).

Executive summary:

Diamminedichloropalladium was tested for its ability to induce reverse mutations in an Ames assay conducted according to OECD Test Guideline 471, using five strains of the bacterium Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537), in the presence or absence of a rat liver metabolic activation (S9) system. Two experiments were conducted, up to the limit of toxicity (126 µg/plate in the absence of S9 and 400 µg/plate in its presence).

Diamminedichloropalladium was not mutagenic in this good-quality Ames assay using five strains of S. typhimurium, when tested up to a limit of toxicity, in the presence and absence of S9.

Reference 2

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:

3 July 2012 - 31 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Growth media (RPMI 1640) prepared from horse serum, penicillin streptomycin, amphotericin B and pluronic.
The mouse lymphoma cells were stored as frozen stocks in liquid nitrogen, purged of mutants and confirmed to be mycoplasma free.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from Aroclor 1254-treated sprague-Dawley rats

Test concentrations with justification for top dose:

1.25, 2.5, 5, 10, 20, 40 ug/mL (range finding experiment)
2.5, 5, 10, 20, 30, 35, 40 ug/mL (assay 1)
5, 10, 20, 25, 30, 35, 40 ug/mL (assay 2)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Diamminedichloropalladium was not soluble in purified water, acetone, ethanol or tetrahydrofuran, but was soluble in DMF. Diamminedichloropalladium solubility in dimethylsulphoxide (DMSO) was not performed at the request of the sponsor as certain chlorinated platinum analogues have been shown to react with DMSO in a manner which was considered likely to impact the scientific integrity of genotoxicity studies.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

4-nitroquinoline-N-oxide without S9 and benzo(a)pyrene with S9.

Details on test system and experimental conditions:

DURATION
- Preincubation period: Not applicable
- Exposure duration: 3 hours incubation at 37+/-1 deg C with gentle agitation.
- Expression time (cells in growth medium): Maintained in flasks for 7 days.
- Selection time (if incubation with a selection agent): Incubated at 37+/-1oC in a humidified incubator gassed with 5+/-1% CO2 v/v in air, for 12 or 13 days.
- Fixation time (start of exposure up to fixation or harvest of cells): 19 to 20 days.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S-9, was in duplicate.

NUMBER OF CELLS EVALUATED: 384 wells at 2X10E+4 cells per well.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage relative survival (% RS).

OTHER EXAMINATIONS:
- Determination of mutation frequency

Evaluation criteria:

For valid data the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative contro (p 2. There was a significant concentration-relationship as indicated by the linear trend analysis (p 3. The effects described above were reproducible.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

limited by solubility in the primary vehicle.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant changes in osmolality or pH were observed at the highest concentration tested (40.00 ug/mL) as compared to the concurrent vehicle controls .

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In an OECD Test Guideline 476 study, to GLP, diamminedichloropalladium failed to induce biologically relevant increases in mutations at the hprt locus of mouse lymphoma (L5178Y) cells when tested up to the limits of solubility in two independent experiments, each in the absence and presence of S9

Executive summary:

In a GLP study, conducted according to OECD Test Guideline 476, diamminedichloropalladium was assessed for its potential to induce mutation at the hprt locus in mouse lymphoma (L5178Y) cells.

In a cytotoxicity range finding experiment, concentrations from 1.25 to 40 µg/mL (limited by solubility in the primary vehicle) were tested with and without rat liver S9 activation. The highest concentration tested gave 90% and 129% relative survival with and without S9, respectively.

In experiment 1 and 2, cells were incubated for 3 hours at concentrations of 5 to 40 µg/mL, in the absence and presence of S9. No evidence of toxicity (measured by relative survival) was seen, and no significant increases in mutant frequency (MF) were observed at any concentration, compared to the concurrent vehicle controls.

 

In experiment 1 only, linear trends were observed in MF, with and without S9. However, since there were no significant increases in MF and no significant linear trends present in experiment 2, the linear trends in experiment 1 were not considered biologically relevant. Under the conditions of this study, diamminedichloropalladium did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells, in the presence and absence of S9, and hence can be considered negative in this assay

Reference 3

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:

17 January 2012 (initiation) - 19 March 2012 (study completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP, with very minor deviations relating to pH measurement that would not affect the study integrity or the validity of the result.

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487 (In vitro mammalian cell micronucleus test)

Deviations:

yes

Remarks:

Very minor deviation relating to pH measurement that would not affect the study integrity or the validity of the result.

Principles of method if other than guideline:

The test methodology is based on OECD guideline 487 (OECD, 2010) and accepted scientific/regulatory principles described in current guidelines for clastogenicity testing in vitro (Fenech, 1998; Fenech et al., 2003; Rosefort et al., 2004; Elhajouji et al., 1998; Migliore & Nieri, 1991; Galloway et al., 1994; Aardema et al., 1998; Miller et al., 1998; ICH-S2A, 1995; ICH-S2B, 1997; Fenech et al., 1999; Thybaud et al., 2007).

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Pooled blood from two healthy, non-smoking female volunteers (aged 28 and 30) was used to prepare duplicate human lymphocyte cultures. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from male Sprague Dawley rats induced with Aroclor 1254 (MolTox (TM))

Test concentrations with justification for top dose:

1.25, 2.5, 5, 10, 20, 40 ug/mL (range finding experiment)
2.5, 5, 10, 20, 30, 35, 40 ug/mL (assay 1)
5, 10, 20, 25, 30, 35, 40 ug/mL (assay 2)

Vehicle / solvent:

DMF (dimethylformamide)
Justification for choice of vehicle: Diamminedichloropalladium was not soluble in purified water, acetone, ethanol or tetrahydrofuran, but was in DMF. Diamminedichloropalladium solubility in dimethylsulphoxide (DMSO) was not performed at the request of the sponsor as certain chlorinated platinum analogues have been shown to react with DMSO in a manner which was considered likely to impact the scientific integrity of genotoxicity studies

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC, clastogenic) or Vinblastine (VIN, aneugenic) in the absence of S9; cyclophosphamide (CPA, clastogenic) in the presence of S9

Remarks:

Cells receiving the positive control substances were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment; All controls were considered valid.

Details on test system and experimental conditions:

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 3 hours in the presence and absence of S9; also 24 hours in the absence of S9.
- Recovery time: 21 hours in the presence and absence of S9; also 24 hours in the absence of S9, following 24-hour treatment.

FIXATIVE: Cells were fixed by dropping a potassium chloride suspension of swollen cells into fresh, cold methanol/glacial acetic acid (3:1, v/v)
STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline (PBS), pH 6.8

NUMBER OF CELLS EVALUATED: Proportions of mono-, bi- and multinucleate cells were evaluated for a minimum of 500 cells per culture. One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.

EVALUATION PROCEDURE: The number of cells containing micronuclei and the number of micronuclei per cell on each slide was noted. Observations were recorded on raw data sheets. The microscope stage co ordinates of the first six micronucleated cells were recorded.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity (%) = (100- relative replication index)

OTHER DETAILS ON TEST SYSTEM:
Test article solutions were protected from light and used within approximately 4 hours of initial formulation.

Vehicle controls were performed in quadruplicate; untreated controls, test article preparations and positive controls were performed in duplicate.

From the range-finder experiment, at least 200 cells per concentration were evaluated for replication index, and cytotoxicity was calculated from this to enable the selection of a suitable range of concentrations for the main micronucleus experiment.

For each treatment regime, two vehicle control cultures were analysed for micronuclei. Positive control concentrations, which gave satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, were analysed.

Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Binucleate (BN) cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.

A micronucleus (MN) was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

Statistics:

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p less-than-or-equal-to 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Up to 5% cytotoxicity in the 24-hour exposure regime in the absence of S9 only; not observed in the other two exposure regimes (but tested upto limit of solubility)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment of cells with diamminedichloropalladiumin the absence and presence of S9 resulted in frequencies of MNBN cells which were similar to and not significantly different from those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all but one diamminedichloropalladium-treated culture fell within normal ranges. The exception occurred in a single culture at the intermediate concentration following 3+21 hour treatment in the absence of S9 (1.1% compared to the normal range of 0.1-1.0%). This marginal increase was not reproduced in the replicate culture at this concentration or at any other concentration analysed in any treatment condition; therefore, it can be considered of no biological relevance.

No significant changes in osmolality or pH were observed at the highest concentration tested (40 ug/mL) as compared to the concurrent vehicle controls

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In an in vitro mammalian cell micronucleus test, conducted according to GLP and OECD Test Guideline 487, diamminedichloropalladium (at up to 40 µg/ml; limited by solubility) did not cause a treatment-related increase in the frequency of micronuclei in cultured human peripheral blood lymphocytes, with or without S9

Executive summary:

The genotoxicity of diamminedichloropalladium has been investigated in a reliable in vitro mammalian cell micronucleus test, conducted to GLP and according to OECD Test Guideline 487. Following a range-finding study, whole blood was obtained from two healthy non-smoking female volunteers. Blood was treated with the test substance (in dimethylformamide) at up to 40 µg/ml (limited by solubility in the vehicle), with or without rat liver S9. Treatment was either continuous (24 hours, without S9 only), or for 3 hours (with and without S9), followed by a 21-hour recovery phase.

No statistically significant increase in the frequency of micronucleated lymphocytes was seen. Positive and negative controls performed as expected.

Under the conditions of this assay, diamminedichloropalladium displayed no evidence of chromosome damage (micronuclei formation) in human lymphocytes, when tested up to the limit of solubility, with or without the addition of S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

Diamminedichloropalladium was tested for its ability to induce reverse mutations in an Ames assay conducted according to OECD Test Guideline 471, using five strains of the bacterium Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537), in the presence or absence of a rat liver metabolic activation (S9) system. Two experiments were conducted, up to the limit of toxicity (126 µg/plate in the absence of S9 and 400 µg/plate in its presence). Diamminedichloropalladium was not mutagenic in this good-quality Ames assay using five strains of S. typhimurium, when tested up to a limit of toxicity, in the presence and absence of S9 (McGarry, 2012a). In support, diamminedichloropalladium was not mutagenic in the preliminary screening Ames assay using three S. typhimurium strains (TA98, TA100 and TA102), when tested at up to cytotoxic concentrations in the presence and absence of S9 (McGarry, 2012b). No activity was seen in a limited Ames test, in four strains of S. typhimurium (TA98, TA100, TA1535 and TA1538) when tested at up to 1 mg/plate, in the absence of S9 (Suraikina et al., 1979). [Testing in the absence of metabolic activation is not considered a critical deficiency for inorganics.]

In a GLP study, conducted according to OECD Test Guideline 476, diamminedichloropalladium was assessed for its potential to induce mutation at the hprt locus in mouse lymphoma (L5178Y) cells. In a cytotoxicity range finding experiment, concentrations from 1.25 to 40 µg/mL (limited by solubility in the primary vehicle) were tested with and without rat liver S9 activation. The highest concentration tested gave 90% and 129% relative survival with and without S9, respectively. In experiment 1 and 2, cells were incubated for 3 hours at concentrations of 5 to 40 µg/mL, in the absence and presence of S9. No evidence of toxicity (measured by relative survival) was seen, and no significant increases in mutant frequency (MF) were observed at any concentration, compared to the concurrent vehicle controls. In experiment 1 only, linear trends were observed in MF, with and without S9. However, since there were no significant increases in MF and no significant linear trends present in experiment 2, the linear trends in experiment 1 were not considered biologically relevant. Under the conditions of this study, diamminedichloropalladium did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells, in the presence and absence of S9, and hence can be considered negative in this assay (Lloyd, 2013).

The genotoxicity of diamminedichloropalladium has been investigated in a reliable in vitro mammalian cell micronucleus test, conducted to GLP and according to OECD Test Guideline 487. Following a range-finding study, whole blood was obtained from two healthy non-smoking female volunteers. Blood was treated with the test substance (in dimethylformamide) at up to 40 µg/mL (limited by solubility in the vehicle), with or without rat liver S9. Treatment was either continuous (24 hours, without S9 only), or for 3 hours (with and without S9), followed by a 21-hour recovery phase. No statistically significant increase in the frequency of micronucleated lymphocytes was seen. Positive and negative controls performed as expected. Under the conditions of this assay, diamminedichloropalladium displayed no evidence of chromosome damage (micronuclei formation) in human lymphocytes, when tested up to the limit of solubility, with or without the addition of S9 (Watters, 2012). In support, in a limited study, the ability of diamminedichloropalladium (in DMSO) to induce micronuclei in human peripheral mononuclear blood cells (lymphocytes) was assessed, in the absence of S9 activation only. The mean numbers of micronuclei in binucleate cells were 8.5, 10.0 and 12.0 at concentrations of 0, 100 and 300 µM, respectively, so treatment produced no statistically significant change from the negative control. At 600 µM, severe cytotoxicity was seen and no assessment of chromosome damage was possible. In conclusion, diamminedichloropalladium did not induce chromosome damage in a limited cytokinesis-block micronucleus test with human lymphocytes that tested only two viable test concentrations, in the absence of S9 (Gebel et al., 1997). [Current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated, with and without S9 (although S9 addition is not considered critical for testing of inorganics).]

In a limited study, the ability of diamminedichloropalladium (at 24-1147 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 382 µM. A maximum induction factor (IFmax, in the absence of cytotoxicity) of 1.43 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).

No in vivo data were identified.

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

Justification for classification or non-classification

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