Diamminedichloropalladium

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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.

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Diamminedichloropalladium

EC Number:

238-269-3

EC Name:

Diamminedichloropalladium

Cas Number:

14323-43-4

Molecular formula:

Cl2H6N2Pd

IUPAC Name:

Diamminedichloropalladium

Details on test material:

- Name of test material (as cited in study report): diamminedichloropalladium
- Substance type: technical product
- Physical state: Solid (light orange powder)
- Analytical purity: >99.5% (Palladium content 50.33%)
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components (in ppm): Pt (<5); Ru (<2); Rh (6); Ir (<10); Au (<2); Ag (<2); Al (<2); Ca (<1); Co (<2); Cr (<1); Cu (3); Fe (<2); Mg (<2); Mn (<5); Ni (<1); Pb (<2); Sb (<10); Si (<10); Sn (<5); Zn (<2)
- Lot/batch No.: 11011
- Stability under test conditions: No data
- Storage condition of test material: 15-30 degrees C, protected from light
- Received: 12 January 2012
- Expiry date: 31 October 2012

Method

Target gene:

Histidine operon

Species / strain

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.

Controls

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).

Results and discussion

Test results

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'.

Applicant's summary and conclusion

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.