Tetraamminepalladium(2+) dichloride

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tetraamminepalladium diacetate

IUPAC Name:

Tetraamminepalladium diacetate

Constituent 2

Reference substance name:

Tetraamminepalladium(2+) diacetate

EC Number:

262-819-1

EC Name:

Tetraamminepalladium(2+) diacetate

Cas Number:

61495-96-3

Molecular formula:

C2H3O2.1/2H12N4Pd

IUPAC Name:

Tetraamminepalladium(2+) diacetate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Tetraamminepalladium (II) acetate
- Substance type: No data
- Physical state: Yellow liquid
- Analytical purity: No data
- Impurities (identity and concentrations): Chlorine (0.022%). Less than 100 ppm calcium, and less than 50 ppm gold, copper, iron, iridium, magnesioum, lead, platinum, rhodium ruthenium and silicon are reported in the certificate of analysis.
- Composition of test material, percentage of components: Tetraamminepalladium(II) acetate solution (44.16% w/w/; 16.06% w/w palladium content)
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: 7300/34-10
- Expiration date of the lot/batch: Retest date: July 2015
- Stability under test conditions: No data
- Storage condition of test material: 15-25 deg C in a sealed container, protected from direct sunlight

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.