Disodium tetrachloropalladate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an OECD Test Guideline 471 study, to GLP, dihydrogen tetrachloropalladate (solution) failed to induce an increase in mutation frequency in four S. typhimurium strains or in E. coli WP2 uvrA, either with or without S9, when tested at up to the limits of cytotoxicity (Verspeek-Rip, 2002).

 

In an OECD Test Guideline 476 study, to GLP, dihydrogen tetrachloropalladate (in solution) did not induce biologically relevant increases in mutant frequency at the hprt locus of mouse lymphoma (L5178Y ) cells when tested up to cytotoxic concentrations in two independent experiments, each in the absence and presence of S9 (Lloyd, 2015a).

 

In an OECD Test Guideline 487 study, to GLP, dihydrogen tetrachloropalladate (in solution) failed to induce biologically significant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+21 hours in the absence and presence of S9 and for 24+0 hours in the absence of S9 (Lloyd, 2015b). In support, in a limited study, dipotassium tetrachloropalladate did not significantly increase the number of micronuclei in human lymphocytes, in the absence of S9 (Gebel et al., 1997).

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:

10 October 2002 to 25 October 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, EEC), to GLP, on closely-related surrogate

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9, microsomal fraction derived from Aroclor 1254-induced rat liver. The S9 mix contained 5% (v/v) S9 fraction in the first two studies and 10% (v/v) in the third study.

Test concentrations with justification for top dose:

Study 1:
3, 10, 33, 100, 333, 1000, 3300 and 5000 μg/plate for TA100 and WP2 uvrA.

Study 2:
3, 10, 33, 100, 200 and 333 μg/plate for TA15335, TA1537 and TA98.

Study 3:
3, 10, 33, 100 and 200 μg/plate for TA15335, TA1537 and TA98 without S9;
10, 33, 100, 200 and 333 μg/plate for TA15335, TA1537 and TA98 with S9;
10, 33, 100, 200 and333 μg/plate for TA100 and WP2 uvrA with or without S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

650 μg/plate for TA100 without S9

Positive control substance:

sodium azide

Remarks:

5 μg/plate for TA1535 without S9

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

10 μg/plate for WP2 uvrA without S9

Positive control substance:

9-aminoacridine

Remarks:

60 μg/plate for TA1537 without S9

Positive control substance:

other: daunomycin

Remarks:

4 μg/plate for TA98 without S9

Positive control substance:

other: 2-aminoanthracene

Remarks:

With 5% S9: 1 μg/plate for TA1535, TA98 and TA100; 2.5 μg/plate for TA1537; 5 μg/plate for WP2 uvrA. With 10% S9: 2.5 μg/plate for TA1535, TA1537, TA98 and TA100; 10 μg/plate for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 hr

NUMBER OF REPLICATIONS: Plating done in triplicate. Each bacterial strain tested in two independent studies

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test substance was considered to be mutagenic if the number of revertant colonies was at least twice that of the spontaneous revertants and reproducible in at least one independently repeated experiment. However any mean plate count of less than 20 revertants was considered to be not significant.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: yes. In the range-finding study, dihydrogen tetrachloropalladate (II)-solution was tested (in triplicate) at up to 5 mg/plate, in the presence or absence of a 5% rat liver metabolic activiation (S9) system. Cytotoxicity was seen at concentrations of 0.33 mg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA: yes and acceptable minimum and maximum numbers of spontaneous revertants and revertants induced by the positive controls given in the report.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Interpretation of results (migrated information):
negative

In an OECD Test Guideline 471 study, to GLP, dihydrogen tetrachloropalladate (solution) failed to induce an increase in mutation frequency in four S. typhimurium strains or in E. coli WP2 uvrA, either with or without S9, when tested at up to the limits of cytotoxicity.

Executive summary:

The mutagenic potential of dihydrogen tetrachloropalladate (solution) was assessed in a reverse mutagenicity assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assessed in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and in Escherichia coli WP2 uvrA, in an attempt to detect both base-pair substitution and frameshift mutations.

In the range-finding study, dihydrogen tetrachloropalladate was tested (in triplicate) at up to 5 mg/plate, in the presence or absence of a 5% rat liver metabolic activation (S9) system. Cytotoxicity was seen at concentrations of 0.33 mg/plate and above. In Salmonella strains TA1535, TA1537 and TA98, cytotoxicity was observed at doses of 0.2 and 0.33 mg/plate, without or with a 5% rat liver S9 respectively. In an independent repeat study using all five strains, but this time without or with a 10% S9 fraction in the S9 mix, cytotoxicity was again seen at 0.2 and 0.33 mg/plate respectively.

Dihydrogen tetrachloropalladate (solution) did not cause an increase in mutation frequency, either with or without metabolic S9 activation, when compared to the spontaneous mutation frequency. In contrast, the known mutagens used as positive controls showed the expected mutagenic activity.

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:

17 November 2014 - 19 February 2015 (experimental)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP, on closely-related surrogate

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 confirmed to be mycoplasma free

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

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: 78.22, 156.4, 312.9, 625.8, 1252 and 2503 μg/mL (both with and without S9)
Experiment 1: 25, 50, 100, 150, 175, 200, 225, 250, 280, 320, 380 and 450 μg/mL (without S9) and 25, 50, 100, 150, 200, 250, 280, 320, 360, 420, 500 and 600 μg/mL (with S9)
Experiment 2: 50, 100, 150, 180, 210, 240, 260, 280, 300, 350 and 400 μg/mL (without S9) and 50, 100, 150, 200, 240, 280, 320, 340, 360, 400 and 450 μg/mL (with S9)

Calculation of all test article concentrations stated in this report include a correction for purity using a factor of 1.908, to allow for the Dihydrogen tetrachloropalladate (II) content of 52.41%.

Vehicle / solvent:

Purified water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Comprised treatments with the vehicle purified water diluted 10-fold in the treatment medium

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 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): 13-14 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); and c) the effects were reproducible. The test article was considered negative in this assay if none of these three criteria were met.

Statistics:

Dunnett's test (one-sided) was used for the analysis of the statistical significance (significant at 5% level) of increased mutant frequencies at each different concentration relative to control.

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 320-450 μg/mL (Exp 1, without S9); 420-600 μg/mL (Exp 1, with S9); 400 μg/mL (Exp 2, without S9) and 450 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 were observed in the range-finder experiment at the highest concentration analysed (1252 µg/mL) in the absence and presence of S9, compared to the concurrent vehicle controls (individual data not reported). No marked changes in pH (compared to the concurrent vehicle controls) were observed in the range-finder at 1252 µg/mL in the presence of S9, but a decrease in pH of >1 unit was observed at 1252 µg/mL in the absence of S9 (see table 1). No further measurements were made in Experiments 1 and 2.

In the cytotoxicity range-finder experiment, the highest concentrations to give a relative survival percentage of at least 10% were 156.4 μg/mL (without S9) and 312.9 μg/mL (with S9) (see table 2).

In Experiment 1, precipitation occurred following the 3-hr treatment period in the highest concentration tested in the presence of S9 (600 μg/mL). Seven days after treatment, the highest three concentrations in the absence (320 to 450 μg/mL) or presence (420 to 600 μg/mL) of S9 were considered too toxic for selection to determine viability and 6TG resistance (see table 3). In Experiment 2, no precipitate was observed in the absence or presence of S9. Seven days after treatment, the highest concentrations tested in the absence and presence of S9 (400 and 450 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 (360 and 400 μg/mL, giving 38% and 13% relative survival (RS), respectively) and at one intermediate concentration (240 μg/mL, giving 59% RS) but there was no statistically significant linear trend. The mean MF values at 240, 360 and 400 μg/mL were 5.35, 4.90 and 4.26 mutants/10^6 viable cells, respectively, compared to the concurrent vehicle control MF value of 1.09. At the time of Experiment 2, the historical mean vehicle control MF value was 3.17, therefore any vehicle control value below 9.51 (3.17 x 3) would be considered acceptable. The MF values at 240, 360 and 400 μg/mL were only marginally greater than 3.17, but were compared against a low vehicle control MF, therefore the increases over the vehicle control MF were statistically significant despite being small in magnitude. Furthermore, there was no evidence of reproducibility between experiments in the presence of S9 (the vehicle control MF was 4.86 in Experiment 1 in the presence of S9) and no statistically significant linear trends in Experiments 1 (with or without S9) and 2 (without S9), therefore the small, non-reproducible increases seen in Experiment 2 were considered not biologically relevant.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Table 1: pH measurements of stock formulations

Experiment	Concentration of stock formulation (mg/mL)	pH of stock formulation
Range-finder	34.54	0.96
1	6.000	1.42
2	4.500	1.54

Table 2: Cytotoxicity data from range-finding test

Concentration (μg/mL)	Relative survival (%) [without S9]	Relative survival (%) [with S9]
0	100	100
78.22	90	80
156.4	108	55
312.9	0	28
625.8	0	0
1252 *	0	0
2503 *	**	**

* 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	3.03	0	100	4.86
25	119	4.83	25	105	1.80
50	102	2.88	50	105	3.11
100	101	2.19	100	118	1.11
150	83	4.87	150	98	0.82
175	77	5.89	200	43	2.11
200	50	2.73	250	35	4.36
225	39	4.25	280	40	3.92
250	29	3.35	320	14	2.73
280	16	2.51	360	16	3.53
NQO 0.15	35	41.83	B[a]P 2	66	30.78
NQO 0.20	6	66.62	B[a]P 3	46	50.88

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	1.85	0	100	1.09
50	105	3.86	50	80	2.45
100	91	3.73	100	96	2.09
150	89	2.70	150	91	3.22
180	83	3.34	200	72	2.69
210	67	4.51	240	59	5.35
240	66	3.01	280	51	1.83
260	38	5.21	320	41	2.91
280	33	4.19	340	32	2.62
300	31	3.51	360	38	4.90
350	16	5.18	400	13	4.26
NQO 0.15	40	30.48	B[a]) 2	79	13.62
NQO 0.20	30	42.28	B[a]P 3	51	50.08

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

Conclusions:

Interpretation of results (migrated information):
negative

In a OECD Test Guideline 476 study, to GLP, dihydrogen tetrachloropalladate (in solution) failed to induce biologically relevant increases in mutant frequency at the hprt locus of mouse lymphoma (L5178Y ) cells when tested up to cytotoxic concentrations in two independent experiments, each in the absence and presence of S9.

Executive summary:

A study was conducted to assess the potential of dihydrogen tetrachloropalladate (in solution) to induce mutations at the hprt locus of L5178Y mouse lymphoma cells. The study was undertaken in compliance with OECD Test Guideline 476 and according to GLP.

Cells were exposed to test material for 3 hr in two independent experiments, each in the absence and presence of a rat liver metabolic activation system (S9). Concentrations of 25 to 450 μg/mL and 25 to 600 μg/mL (Experiment 1, without and with S9, respectively) and 50 to 400 μg/mL and 50 to 450 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 320-450 μg/mL (Experiment 1, without S9); 420-600 μg/mL (Experiment 1, with S9); 400 μg/mL (Experiment 2, without S9) and 450 μg/mL (Experiment 2, with S9) being considered too toxic for selection to determine viability and 6TG resistance.

No statistically significant increases in mutation frequency (MF) over the concurrent vehicle control value were observed in Experiment 1 (with or without S9) or in Experiment 2 in the absence of S9. A statistically significant increase in MF was, however, seen in Experiment 2 in the presence of S9, at the highest two concentrations analysed (380 and 400 μg/mL) and at one intermediate concentration (240 μg/mL). The mean MF values at 240, 380 and 400 μg/mL were 5.35, 4.90 and 4.26 mutants/10^6 viable cells, respectively, compared to the concurrent vehicle control MF value of 1.09. These increases were small in magnitude and only statistically significant because of an unusually low vehicle control value (the historical control value was 3.17). Furthermore, there was no evidence of reproducibility between experiments in the presence of S9 (the vehicle control MF was 4.86 in Experiment 1) and no statistically significant linear trends in Experiments 1 (with or without S9) and 2 (without S9), therefore the small, non-reproducible increases seen in Experiment 2 were considered not biologically relevant.

Overall, dihydrogen tetrachloropalladate failed to induce biologically significant increases in mutation frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic concentrations in two independent experiments, each in the absence and presence of S9.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 September 2014 - 28 October 2014 (experimental)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP, on closely-related surrogate

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 487: In Vitro Mammalian Cell Micronucleus Test

Deviations:

yes

Remarks:

No justication provided for use of alternative (noscapine) positive control.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

N/A

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Blood from four healthy, non-smoking female volunteers (25-35 years of age). Not suspected of any viral infeection or exposed to high levels of radiation or hazardous chemicals. All donors were not heavy drinkers of alcohol, and none were taking any form of medication (excluding the contraceptive pill).

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: 9.081, 15.13, 25.22, 42.04, 70.07, 116.8, 194.6, 324.4, 540.7, 901.1, 1502 and 2503 μg/mL (both with and without S9).
Micronucleus Experiment (3+21 hr): 50, 100, 200, 300, 340, 380, 420, 450, 480, 510, 550, 600, 750 μg/mL (without S9) and 100, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900 μg/mL (with S9). Micronucleus experiment (24+0 hr): 20, 40, 60, 80, 100, 110, 120, 130, 140, 170, 200, 300 μg/mL (without S9).

Calculation of all test article concentrations stated in the study report include a correction for purity using a factor of 1.908, to allow for the Dihydrogen tetrachloropalladate (II) content of 52.41%.

Vehicle / solvent:

Purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Noscapine

Remarks:

Cyclophosphamide (CPA) and noscapine (NOS) dissolved in DMSO immediately prior to use and diluted accordingly. Mitomycin (MMC) dissolved in water prior to use and diluted. No justification for use of NOS as positive control.

Details on test system and experimental conditions:

For each experiment, an appropriate volume of whole blood (from healthy female volunteers) was drawn from the peripheral circulation into heparinsed tubes within one day of culture initation. The measured cell cycle time of the donors used fell within the range 13 +/- 2 hours. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 ml of pooled heparinsed blood into pre-warmed buffered-medium containing 10% heat inactivated foetal calf serum and 0.52% penicillin/streptomycin (8.6 mL for range-finder experiments, 8.1 mL for micronucleus experiment cultures).

The mitogen phytohaemagglutinin (PHA) was included in the culture medium at 2% to stimulate the lymphocytes to divide.

Immediately prior to treatment, all continuous vehicle control and test article treated cultures had 0.1 mL culture medium removed to give a pre-treatment volume of 8.4 mL. All positive control treated cultures had culture medium added (0.8 mL for continuous cultures or 0.9 mL for pulse cultures) to give pre-treatment volumes of 9.3 and 9.4 mL, respectively.
S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article or vehicle control (1.0 mL per culture) or positive controls (0.1 mL per culture).
Cytochalasin B (cyto-B), formulated in DMSO, was added directly (0.1 mL per culture) to all continuous cultures at the time of treatment to give a final concentration of 6 μg/mL per culture. The final culture volume was 10 mL. Cultures were incubated at 37 +/- 1°C for the designated exposure time (3+21 or 24+0 hr, i.e. hours of treatment + hours recovery).

For removal of the test article, cells were pelleted (approximately 300 x g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37 +/- 1 oC) and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. At the appropriate times, Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 μg/mL per culture.

HARVESTING
At the defined sampling time, cultures were centrifuged at approximately 300 x g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl at 37 +/- 1 oC for 4 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation (approximately 300 x g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 x g, 2-3 minutes) until the cell pellets were clean.

1,000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. Nucleoplasmic bridges between nuclei and binucleate cells were recorded during the micronucleus experiments to provide an indication of chromosome rearrangement. After scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.

Cytotoxicity was expressed as calculated from the replication index (RI), which indicates the relative number of nuclei compared to vehicle controls. Cytotoxicity = 100 - relative RI (%).

No marked changes in osmality were observed in the range-finder at the highest concentration (2503 μg/mL) tested in the absence and presence of S9, compared to the vehicle controls.

Marked reductions in pH (of >1 pH unit, compared to the concurrent vehicle control) were observed in post-treatment media in the range-finder at the highest concentration (2503 μg/mL) for 3+21 hours in the absence of S9 and at the highest two concentrations (1502 and 2503 μg/mL) tested for 3+21 hours with S9. However, the pH values at 901.1 μg/mL were within 1 pH unit of the vehicle control values under all treatment conditions tested in the range-finder and no concentrations greater than 900 μg/mL were tested in the micronucleus experiment.

Evaluation criteria:

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

A micronucleus 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.

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 (micronucleated binucleate) 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.

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 proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test. Probability values of p=<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:

concentrations of 600-750 μg/mL (3+21 hr without S9), 700-900 μg/mL (3+21 hr with S9), and 120-300 μg/mL (24+0 treatment without S9) were considered too cytotoxic for selection (i.e. RI >60%).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No marked changes in osmolality and pH were observed in the Micronucleus Experiment at the highest concentrations tested under all treatment conditions, compared to the concurrent vehicle controls.
Treatment of cells with Dihydrogen tetrachloropalladate (II) for 3+21 hours in the absence and presence of S9 resulted in frequencies of MNBN cells that were similar to and not significantly higher than the concurrent vehicle controls at all concentrations analysed. The MNBN cell frequencies of all Dihydrogen tetrachloropalladate (II) treated cultures under both treatment conditions fell within the historical control ranges and there was no indication of a concentration related response.
Treatment of cells for 24+0 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p=<0.05) than the concurrent vehicle controls at the highest concentration analysed (110.0 µg/mL, giving a 59% reduction in RI). However, the MNBN cell frequencies of all treated cultures fell within the historical control range and the MNBN cell frequencies of cultures analysed at 100.0 µg/mL, giving 48% reduction in RI (i.e. very close to the optimum toxicity level of 50-60%), were very similar to (and not significantly higher than) the concurrent vehicle control value. The small but statistically significant increase in MNBN cell frequency seen at 110.0 µg/mL was therefore seen only at the limit of cytotoxicity and was considered not to be biologically relevant.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Interpretation of results (migrated information):
negative

In a OECD Test Guideline 487 study, to GLP, dihydrogen tetrachloropalladate (in solution) failed to induce biologically significant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+21 hours in the absence and presence of S9 and for 24+0 hours in the absence of S9.

Executive summary:

A study was conducted to evaluate the clastogenic and aneugenic potential of dihydrogen tetrachloropalladate (in solution) by examining its effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated in the absence and presence of a rat liver metabolising (S9) system. The study was undertaken in compliance with OECD Test Guideline 487 and according to GLP.

Treatments covering (20-900 µg/mL) a broad range of concentrations, separated by narrow intervals, were performed with and without S9. The test article was formulated in purified water. The highest concentrations tested in the Micronucleus experiment were limited by toxicity and were determined following a preliminary cytotoxicity range-finder experiment. Treatments were conducted 48 hr following mitogen stimulation by PHA. The test article concentrations for micronucleus analysis were selected by evaluating the effect of dihydrogen tetrachloropalladate on the replication index. Micronuclei were analysed at four concentrations.

Frequencies of micronucleated binucleate cells resulting from treatment with dihydrogen tetrachloropalladate (3 + 21 hr, with and without S9) were similar to and not significantly higher than the concurrent vehicle controls at all concentrations analysed. The frequencies under both treatment conditions were within the historical control ranges and there was no indication of a concentration-related response. Treatment of cells with dihydrogen tetrachloropalladate (24 + 0 hours in the absence of S9) resulted in frequencies of micronucleated binucleate (MNBN) cells that were significantly higher than the concurrent vehicle controls at 110 µg/mL (the highest concentration analysed). However, the MNBN cell frequencies of all treated cultures fell within the normal range and the small but statistically significant increase in MNBN cell frequency observed was therefore seen only at the limit of toxicity and was considered not biologically relevant.

Dihydrogen tetrachloropalladate (in solution) did not induce biologically significant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3 + 21 hours in the absence and presence of S9 and for 24 + 0 hours in the absence of S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo data were identified.

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).

 

The mutagenic potential of dihydrogen tetrachloropalladate (in solution) was assessed in a reverse mutagenicity assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assessed in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and in Escherichia coli WP2 uvrA, in an attempt to detect both base-pair substitution and frameshift mutations. In the range-finding study, dihydrogen tetrachloropalladate (in solution) was tested (in triplicate) at up to 5 mg/plate, in the presence and absence of a 5% rat liver metabolic activation (S9) system. Cytotoxicity was seen at concentrations of 0.33 mg/plate and above. In Salmonella strains TA1535, TA1537 and TA98, cytotoxicity was observed at doses of 0.2 and 0.33 mg/plate, without or with a 5% rat liver S9 respectively. In an independent repeat study using all five strains, but this time without or with a 10% S9 fraction in the S9 mix, cytotoxicity was again seen at 0.2 and 0.33 mg/plate respectively. Dihydrogen tetrachloropalladate (in solution) did not cause an increase in mutation frequency, either with or without metabolic S9 activation, when compared to the spontaneous mutation frequency. In contrast, the known mutagens used as positive controls showed the expected mutagenic activity (Verspeek-Rip, 2002).

 

In support, in limited Ames assays, dipotassium tetrachloropalladate was not mutagenic in two strains of S. typhimurium (TA98 and TA100), when tested at up to 100 nmol/plate in the absence and presence of S9 (Uno and Morita, 1993) or at up to 1 mg/plate in the absence of S9 only (Suraikina et al., 1979).

 

A study was conducted to assess the potential of dihydrogen tetrachloropalladate (in solution) to induce mutations at the hprt locus of L5178Y mouse lymphoma cells. The study was undertaken in compliance with OECD Test Guideline 476 and according to GLP. Cells were exposed to test material for 3 hr in two independent experiments, each in the absence and presence of a rat liver metabolic activation system (S9). Concentrations of 25 to 450 μg/mL and 25 to 600 μg/mL (Experiment 1, without and with S9, respectively) and 50 to 400 μg/mL and 50 to 450 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 320-450 μg/mL (Experiment 1, without S9); 420-600 μg/mL (Experiment 1, with S9); 400 μg/mL (Experiment 2, without S9) and 450 μg/mL (Experiment 2, with S9) being considered too toxic for selection to determine viability and 6TG resistance. No statistically significant increases in mutation frequency (MF) over the concurrent vehicle control value were observed in Experiment 1 (with or without S9) or in Experiment 2 in the absence of S9. A statistically significant increase in MF was, however, seen in Experiment 2 in the presence of S9, at the highest two concentrations analysed (380 and 400 μg/mL) and at one intermediate concentration (240 μg/mL). The mean MF values at 240, 380 and 400 μg/mL were 5.35, 4.90 and 4.26 mutants/10^6 viable cells, respectively, compared to the concurrent vehicle control MF value of 1.09. These increases were small in magnitude and only statistically significant because of an unusually low vehicle control value (the historical control value was 3.17). Furthermore, there was no evidence of reproducibility between experiments in the presence of S9 (the vehicle control MF was 4.86 in Experiment 1) and no statistically significant linear trends in Experiments 1 (with or without S9) and 2 (without S9), therefore the small, non-reproducible increases seen in Experiment 2 were considered not biologically relevant. Overall, dihydrogen tetrachloropalladate (in solution) failed to induce biologically significant increases in mutation frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic concentrations in two independent experiments, each in the absence and presence of S9 (Lloyd, 2015a).

 

A study was conducted to evaluate the clastogenic and aneugenic potential of dihydrogen tetrachloropalladate (in solution) by examining its effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated in the absence and presence of a rat liver metabolising (S9) system. The study was undertaken in compliance with OECD Test Guideline 487 and according to GLP. Treatments covering (20–900 µg/mL) a broad range of concentrations, separated by narrow intervals, were performed with and without S9. The test article was formulated in purified water. The highest concentrations tested in the micronucleus experiment were limited by toxicity and were determined following a preliminary cytotoxicity range-finder experiment. Treatments were conducted 48 hr following mitogen stimulation by PHA. The test article concentrations for micronucleus analysis were selected by evaluating the effect of dihydrogen tetrachloropalladate (in solution) on the replication index. Micronuclei were analysed at four concentrations. Frequencies of micronucleated binucleate cells resulting from treatment with dihydrogen tetrachloropalladate (3+21 hr, with and without S9) were similar to and not significantly higher than the concurrent vehicle controls at all concentrations analysed. The frequencies under both treatment conditions were within the historical control ranges and there was no indication of a concentration-related response. Treatment of cells with dihydrogen tetrachloropalladate (24+0 hours in the absence of S9) resulted in frequencies of micronucleated binucleate (MNBN) cells that were significantly higher than the concurrent vehicle controls at 110 µg/mL (the highest concentration analysed). However, the MNBN cell frequencies of all treated cultures fell within the normal range and the small but statistically significant increase in MNBN cell frequency observed was therefore seen only at the limit of toxicity and was considered not biologically relevant. Dihydrogen tetrachloropalladate (in solution) did not induce biologically significant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+21 hours in the absence and presence of S9 and for 24+0 hours in the absence of S9 (Lloyd, 2015b).

 

In support, in a limited study, dipotassium tetrachloropalladate did not significantly increase the number of micronuclei in human lymphocytes, in the absence of S9 (Gebel et al., 1997).

 

In a limited study, dipotassium tetrachloropalladate (at cytotoxic concentrations) did not induce DNA damage in a bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9 (Gebel et al., 1997).

 

Dihydrogen and dipotassium tetrachloropalladates are considered to fall within the scope of the read-across category "tetrachloropalladate salts". See IUCLID section 13 for full read-across justification report.

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

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No evidence of genotoxic activity has been seen (for read-across compounds) in reliable in vitro assays in bacterial or somatic cells, including GLP guideline studies assessing mutagenic and clastogenic activity. No studies specifically assessing mutagenic potential 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 disodium tetrachloropalladate for germ cell mutagenicity is not warranted, according to EU CLP criteria (EC 1272/2008).