Dipotassium hexachloropalladate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dipotassium hexachloropalladate was not mutagenic in a bacterial reverse mutation (Ames) assay using four Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102), when tested at up to cytotoxic concentrations in the presence and absence of a rat liver metabolic activation (S9) system (Bunger et al., 1996). In an in vitro mouse lymphoma assay, conducted according to GLP and OECD Test Guideline 476, diammonium hexachloropalladate failed to induce mutations when tested up to the limit of solubility (11 µg/ml) in the presence and absence of S9 (Lloyd, 2014a). In an in vitro mammalian cell micronucleus test, conducted according to GLP and OECD Test Guideline 487, diammonium hexachloropalladate at up to the limit of solubility (11 µg/ml) did not cause a treatment-related increase in the frequency of micronuclei in cultured human lymphocytes, with or without S9 (Lloyd, 2014b).

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:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD guidelines. However, only four bacterial strains were tested (TA 1535 omitted).

Qualifier:

according to guideline

Guideline:

other: Revised test protocol of Maron and Ames (1983)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

he study differed principally from OECD TG471 in that only four bacterial strains were tested. The recommended strain TA1535 was ommitted.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

Sprague-Dawley rat liver, induced with Phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

The test substance was dissolved in distilled water and diluted to 5-500 ug/plate [or possibly 10, 50, 100 or 500 ug/plate] in all four tester strains, in the absence or presence of (4% and 10 %) S9. The number of revertant colonies on the plates were recorded after 48 hours of incubation in the dark at 37degC.

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Details on test system and experimental conditions:

The tests were done in duplicate and repeated at least 3 times.

Evaluation criteria:

For the test substance to be considered mutagenic, a two-fold (or more) increase in the mean revertant numbers must be observed in the plates containing the test substance compared to the spontaneous reversion rate.

Statistics:

None used

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100 and TA102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100 and TA102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Additional information on results:

The test substance caused no increase in revertants in any of the four tester strains, in the presence and absence of S9.

"High doses of the metal compounds proved toxic to the tester strains", resulting in a thinning of the background bacterial lawn. Although no actual data were provided for potassium hexachloropalladate, the minimum toxic dose for the palladium salts was apparently 100 ug/plate.

Conclusions:

Interpretation of results (migrated information):
negative

Dipotassium hexachloropalladate was not mutagenic in a bacterial reverse mutation (Ames) assay using four Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102), when tested at up to cyctotoxic concentrations in the presence and absence of a rat liver metabolic activation (S9) system.

Executive summary:

Dipotassium hexachloropalladate was assessed for mutagenic activity in a bacterial reverse mutation (Ames) assay, similar to OECD Test Guideline 471, using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 and tested in the presence and absence of a metabolic activation system (S9) derived from phenobarbital- and beta-naphthoflavone-induced rat livers. (The recommended strain TA1535 was omitted.)

No mutagenic effect was seen in any of the four strains at up to cyctotoxic concentrations, either in the presence or absence of a rat liver metabolic activation (S9) system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

Conducted before requirement for GLP.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

without

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Distilled water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Remarks:

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

Evaluation criteria:

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

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

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

Conclusions:

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

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

Executive summary:

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

Reference 3

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 August to 30 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (main study (Experiments 1 and 2))

Additional strain / cell type characteristics:

other: histidine-requiring

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100 and TA 102 (range-finding study)

Additional strain / cell type characteristics:

other: histidine-requiring

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Range-finding experiment: 0, 0.11, 0.35, 1.1, 3.5, 11.0, 34.8 or 110.0 µg/plate

Main study:
Experiment 1: 0, 0.11, 0.35, 1.1, 3.5, 11.0, 34.8 or 110.0 µg/plate
Experiment 2: 0, 1.7, 3.4, 6.9, 13.8, 27.5, 55.0 or 110.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: not soluble in several commonly used vehicles (including water, acetone, ethanol and tetrahydrofuran)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

benzo(a)pyrene

Remarks:

TA98, with S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

50 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate

Positive control substance:

mitomycin C

Remarks:

TA102, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate(TA100, TA1535, TA1537), 20 µg/plate (TA102)

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA102, TA1535 and TA1537, with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min (Experiment 2, with S-9)
- Exposure duration: 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): ~3 days

NUMBER OF REPLICATIONS: single test plates (range-finding study); triplicate (main study (Experiments 1 and 2))

SELECTION AGENT (mutation assays): histidine-free medium

DETERMINATION OF CYTOTOXICITY
- Method: other: background lawns examined for signs of toxicity (e.g. marked reduction in revertants compared to controls)

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p≤0.01) which was concentration related.
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:

Dunnett's test was used to assess the probability of the observed results arising by chance. Results were considered statistically significant when p≤0.01.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 34.79 and/or 110 µg/plate in all strains, with and without S-9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 55 and/or 110 µg/plate in all strains, with and without S-9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitation observed following incubation
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: cytotoxicity seen from 34.79 and/or 110 µg/plate in all strains tested (TA98, TA100 and TA102), with and without S-9. Range-finding data were considered to be acceptable for cytotoxicity assessment only.

COMPARISON WITH HISTORICAL CONTROL DATA: results for vehicle controls were compared to historical control data from within the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in each case, cytotoxicity ranged from a slight thinning of the background bacterial lawn (with or without a concurrent reduction in revertant number) to a complete killing of the test bacteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment 1

It should be noted that no data were obtained for a single replicate at 110 µg/plate following strain TA1535 treatments in the presence of S-9 in Experiment 1 and for two replicates at 55 µg/plate following strain TA100 treatments in the absence of S-9 in Experiment 2. However, as data for the remaining replicates were obtained at these concentrations, and data were available from all remaining concentrations for these strains in Experiment 1 and Experiment 2 it is considered that there are sufficient data available for mutation assessment and this has not affected the integrity of the assay.

Conclusions:

Interpretation of results (migrated information):
negative

In a good-quality Ames assay, conducted according to GLP and OECD Test Guideline 471, diammonium hexachloropalladate lacked evidence of mutagenic potential in all five Salmonella strains tested (including TA102), at up to a cytotoxic concentration of 110 µg/plate, in the presence and absence of a rat liver metabolic activation (S9) system.

Executive summary:

Diammonium hexachloropalladate has been assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Test Guideline 471. Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were tested with and without the addition of a mammalian (rat liver) metabolic activation (S9) system in two separate experiments. In the first experiment, agar containing the test substance at up to 110 µg/plate was incubated with the bacterial strains for 3 days. The second experiment, also using concentrations of up to 110 µg/plate, included an additional 20-minute pre-incubation step for cultures treated in the presence of S9.

No evidence of mutagenicity was observed in any experiment. Cytotoxicity was observed from 34.79 and/or 110 µg/plate in all strains, with and without S9. In the second experiment, cytotoxicity was seen from 55 and/or 110 µg/plate. Vehicle and positive controls performed as expected. Under the conditions of this assay, diammonium hexachloropalladate was not mutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

27 August to 30 September 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (main study (Experiments 1 and 2))

Additional strain / cell type characteristics:

other: histidine-requiring

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100 and TA 102 (range-finding study)

Additional strain / cell type characteristics:

other: histidine-requiring

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Range-finding experiment: 0, 0.11, 0.35, 1.1, 3.5, 11.0, 34.8 or 110.0 µg/plate

Main study:
Experiment 1: 0, 0.11, 0.35, 1.1, 3.5, 11.0, 34.8 or 110.0 µg/plate
Experiment 2: 0, 1.7, 3.4, 6.9, 13.8, 27.5, 55.0 or 110.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: not soluble in several commonly used vehicles (including water, acetone, ethanol and tetrahydrofuran)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

benzo(a)pyrene

Remarks:

TA98, with S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

50 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate

Positive control substance:

mitomycin C

Remarks:

TA102, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate(TA100, TA1535, TA1537), 20 µg/plate (TA102)

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA102, TA1535 and TA1537, with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min (Experiment 2, with S-9)
- Exposure duration: 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): ~3 days

NUMBER OF REPLICATIONS: single test plates (range-finding study); triplicate (main study (Experiments 1 and 2))

SELECTION AGENT (mutation assays): histidine-free medium

DETERMINATION OF CYTOTOXICITY
- Method: other: background lawns examined for signs of toxicity (e.g. marked reduction in revertants compared to controls)

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p≤0.01) which was concentration related.
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:

Dunnett's test was used to assess the probability of the observed results arising by chance. Results were considered statistically significant when p≤0.01.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 34.79 and/or 110 µg/plate in all strains, with and without S-9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 55 and/or 110 µg/plate in all strains, with and without S-9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitation observed following incubation
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: cytotoxicity seen from 34.79 and/or 110 µg/plate in all strains tested (TA98, TA100 and TA102), with and without S-9. Range-finding data were considered to be acceptable for cytotoxicity assessment only.

COMPARISON WITH HISTORICAL CONTROL DATA: results for vehicle controls were compared to historical control data from within the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in each case, cytotoxicity ranged from a slight thinning of the background bacterial lawn (with or without a concurrent reduction in revertant number) to a complete killing of the test bacteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment 1

It should be noted that no data were obtained for a single replicate at 110 µg/plate following strain TA1535 treatments in the presence of S-9 in Experiment 1 and for two replicates at 55 µg/plate following strain TA100 treatments in the absence of S-9 in Experiment 2. However, as data for the remaining replicates were obtained at these concentrations, and data were available from all remaining concentrations for these strains in Experiment 1 and Experiment 2 it is considered that there are sufficient data available for mutation assessment and this has not affected the integrity of the assay.

Conclusions:

Interpretation of results (migrated information):
negative

In a good-quality Ames assay, conducted according to GLP and OECD Test Guideline 471, diammonium hexachloropalladate lacked evidence of mutagenic potential in all five Salmonella strains tested (including TA102), at up to a cytotoxic concentration of 110 µg/plate, in the presence and absence of a rat liver metabolic activation (S9) system.

Executive summary:

Diammonium hexachloropalladate has been assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Test Guideline 471. Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were tested with and without the addition of a mammalian (rat liver) metabolic activation (S9) system in two separate experiments. In the first experiment, agar containing the test substance at up to 110 µg/plate was incubated with the bacterial strains for 3 days. The second experiment, also using concentrations of up to 110 µg/plate, included an additional 20-minute pre-incubation step for cultures treated in the presence of S9.

No evidence of mutagenicity was observed in any experiment. Cytotoxicity was observed from 34.79 and/or 110 µg/plate in all strains, with and without S9. In the second experiment, cytotoxicity was seen from 55 and/or 110 µg/plate. Vehicle and positive controls performed as expected. Under the conditions of this assay, diammonium hexachloropalladate was not mutagenic.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August to 8 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: whole blood (0.4 ml) in HEPES-buffered RPMI medium (9 ml) containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin/streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- Other: Blood taken from two healthy non-smoking male volunteers for each of the range-finding and main experiments. Blood cultures were incubated for about 48 hours at 37±1°C and rocked continuously before treatment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Range-finding study: 0 or ~0.04-11 µg/ml
Main study: 0 or 2-11 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that the test substance was not soluble in purified water, acetone, ethanol or tetrahydrofuran

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

without S-9 (3-hr treatment), at 0.6 or 0.8 µg/ml

Positive control substance:

mitomycin C

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

with S-9 (3-hr treatment), at 6.25 or 12.5 µg/ml

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

without S-9 (24-hr treatment), at 0.02, 0.03 or 0.04 µg/ml

Positive control substance:

other: vinblastine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 0 hours
- Exposure duration: 3 (with and without S-9) or 24 hours (without S-9)
- Expression time (cells in growth medium): 21 (with and without S-9) or 0 hours (without S-9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: range-finding study: at least 200/concentration; main study: 1000/culture, where possible (2000/concentration)

DETERMINATION OF CYTOTOXICITY
- Method: relative replication index

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 micronucleated binucleate (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.
Results which only partially satisfied the above criteria were dealt with on a case by case basis.

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with MNBN cells in each culture were obtained.
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:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no cultures were excluded from analysis based on cytotoxicity. The highest levels of cytotoxicity seen in the main study were for the 24-hr continuous treatment (without S-9), which saw 7, 7 and 9% at 8, 10 and 11 µg/ml, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes in pH were observed at any concentration tested, compared to the concurrent vehicle control
- Effects of osmolality: osmalarity was measured in the range-finding experiment. No marked changes were observed
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitation seen at up to 11 µg/ml (the highest tested concentration)

RANGE-FINDING/SCREENING STUDIES: range-finding study for cytotoxicity used concentrations of up to 11 µg/ml. In the absence of significant levels of cytotoxicity (highest levels were 15 and 32% at 6.6 and 11 µg/ml in the 24-hour continuous treatment (without S-9)), this was chosen as the high dose in the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: results were compared to historical vehicle control data (see Table 1 in "Any other information on results incl. tables" for historical control ranges)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Results of a micronucleus test on primary human lymphocytes

Treatment	Concentration (mg/mL)	Cytotoxicity (%)^$	Mean MNBN cell frequency (%)	Historical Control Range (%)^#	Statistical significance
3+21 hour -S-9	Vehicle^a	-	0.40	0.10–1.00	-
	8.000	1	0.40		NS
	10.00	0	0.50		NS
	11.00	0	0.70		NS
	*MMC, 0.80	ND	15.15		p ≤ 0 .001
3+21 hour +S-9	Vehicle	-	0.40	0.00–1.00	-
	8.000	0	0.35		NS
	10.00	0	0.35		NS
	11.00	0	0.30		NS
	*CPA, 6.25	ND	2.15		p ≤ 0.001
24+0 hour -S-9	Vehicle	-	0.35	0.10–1.10	-
	8.000	7	0.85		p ≤ 0.05
	10.00	7	0.70		NS
	11.00	9	0.30		NS
	*VIN, 0.02	ND	14.94		p ≤ 0.001
^a          Vehicle control was DMF *          Positive control #         95^thpercentile of the observed range $         Based on replication index NS      Not significant ND      Not determined

 

Treatment of cells with diammonium hexachloropalladate for 3 hours in the absence and presence of S-9 and for 24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were generally similar to those observed in concurrent vehicle controls at all concentrations analysed under all three treatment conditions. There was one instance (following treatment at 8.00 µg/ml for 24 hours in the absence of S-9) in which a statistically significant increase (p ≤ 0.05) in MNBN cell frequency was observed. However, the MNBN cell frequency of all diammonium hexachloropalladate-treated cultures at this and all other concentrations tested fell within normal ranges and there was no evidence of a concentration-related response for the 24-hour –S-9 treatment. This isolated observation was therefore considered not biologically relevant.

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of genotoxicity was seen for diammonium hexachloropalladate in a guideline micronucleus test on primary human lymphocytes, with and without S-9. The test was conducted to GLP, and according to OECD Guideline 487.

Executive summary:

The genotoxicity of diammonium hexachloropalladate has been investigated in an in vitro micronucleus test, conducted to GLP and according to OECD Guideline 487.

 

Following a range-finding study, whole blood was obtained from two healthy male volunteers. Blood was treated with the test substance (in dimethylformamide) at up to 11 µg/ml, with or without metabolic activation (rat liver S-9). Treatment was either continuous (24 hours, without S-9 only), or for 3 hours, followed by a 21-hour recovery phase (with and without S-9).

 

The only statistically significant increased frequency of micronucleated lymphocytes was seen for cultures treated with 8 µg/m for 24 hours without S-9. As other cultures at this and higher concentrations demonstrated no such effect, this was not considered biologically relevant. Positive and negative controls performed as expected.

 

Under the conditions of this assay, diammonium hexachloropalladate displayed no significant evidence of genotoxicity in human lymphocytes, with or without metabolic activation.

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Study period:

29 August to 8 October 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: whole blood (0.4 ml) in HEPES-buffered RPMI medium (9 ml) containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin/streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- Other: Blood taken from two healthy non-smoking male volunteers for each of the range-finding and main experiments. Blood cultures were incubated for about 48 hours at 37±1°C and rocked continuously before treatment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Range-finding study: 0 or ~0.04-11 µg/ml
Main study: 0 or 2-11 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that the test substance was not soluble in purified water, acetone, ethanol or tetrahydrofuran

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

without S-9 (3-hr treatment), at 0.6 or 0.8 µg/ml

Positive control substance:

mitomycin C

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

with S-9 (3-hr treatment), at 6.25 or 12.5 µg/ml

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

without S-9 (24-hr treatment), at 0.02, 0.03 or 0.04 µg/ml

Positive control substance:

other: vinblastine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 0 hours
- Exposure duration: 3 (with and without S-9) or 24 hours (without S-9)
- Expression time (cells in growth medium): 21 (with and without S-9) or 0 hours (without S-9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: range-finding study: at least 200/concentration; main study: 1000/culture, where possible (2000/concentration)

DETERMINATION OF CYTOTOXICITY
- Method: relative replication index

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 micronucleated binucleate (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.
Results which only partially satisfied the above criteria were dealt with on a case by case basis.

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with MNBN cells in each culture were obtained.
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:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no cultures were excluded from analysis based on cytotoxicity. The highest levels of cytotoxicity seen in the main study were for the 24-hr continuous treatment (without S-9), which saw 7, 7 and 9% at 8, 10 and 11 µg/ml, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes in pH were observed at any concentration tested, compared to the concurrent vehicle control
- Effects of osmolality: osmalarity was measured in the range-finding experiment. No marked changes were observed
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitation seen at up to 11 µg/ml (the highest tested concentration)

RANGE-FINDING/SCREENING STUDIES: range-finding study for cytotoxicity used concentrations of up to 11 µg/ml. In the absence of significant levels of cytotoxicity (highest levels were 15 and 32% at 6.6 and 11 µg/ml in the 24-hour continuous treatment (without S-9)), this was chosen as the high dose in the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: results were compared to historical vehicle control data (see Table 1 in "Any other information on results incl. tables" for historical control ranges)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Results of a micronucleus test on primary human lymphocytes

Treatment	Concentration (mg/mL)	Cytotoxicity (%)^$	Mean MNBN cell frequency (%)	Historical Control Range (%)^#	Statistical significance
3+21 hour -S-9	Vehicle^a	-	0.40	0.10–1.00	-
	8.000	1	0.40		NS
	10.00	0	0.50		NS
	11.00	0	0.70		NS
	*MMC, 0.80	ND	15.15		p ≤ 0 .001
3+21 hour +S-9	Vehicle	-	0.40	0.00–1.00	-
	8.000	0	0.35		NS
	10.00	0	0.35		NS
	11.00	0	0.30		NS
	*CPA, 6.25	ND	2.15		p ≤ 0.001
24+0 hour -S-9	Vehicle	-	0.35	0.10–1.10	-
	8.000	7	0.85		p ≤ 0.05
	10.00	7	0.70		NS
	11.00	9	0.30		NS
	*VIN, 0.02	ND	14.94		p ≤ 0.001
^a          Vehicle control was DMF *          Positive control #         95^thpercentile of the observed range $         Based on replication index NS      Not significant ND      Not determined

 

Treatment of cells with diammonium hexachloropalladate for 3 hours in the absence and presence of S-9 and for 24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were generally similar to those observed in concurrent vehicle controls at all concentrations analysed under all three treatment conditions. There was one instance (following treatment at 8.00 µg/ml for 24 hours in the absence of S-9) in which a statistically significant increase (p ≤ 0.05) in MNBN cell frequency was observed. However, the MNBN cell frequency of all diammonium hexachloropalladate-treated cultures at this and all other concentrations tested fell within normal ranges and there was no evidence of a concentration-related response for the 24-hour –S-9 treatment. This isolated observation was therefore considered not biologically relevant.

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of genotoxicity was seen for diammonium hexachloropalladate in a guideline micronucleus test on primary human lymphocytes, with and without S-9. The test was conducted to GLP, and according to OECD Guideline 487.

Executive summary:

The genotoxicity of diammonium hexachloropalladate has been investigated in an in vitro micronucleus test, conducted to GLP and according to OECD Guideline 487.

 

Following a range-finding study, whole blood was obtained from two healthy male volunteers. Blood was treated with the test substance (in dimethylformamide) at up to 11 µg/ml, with or without metabolic activation (rat liver S-9). Treatment was either continuous (24 hours, without S-9 only), or for 3 hours, followed by a 21-hour recovery phase (with and without S-9).

 

The only statistically significant increased frequency of micronucleated lymphocytes was seen for cultures treated with 8 µg/m for 24 hours without S-9. As other cultures at this and higher concentrations demonstrated no such effect, this was not considered biologically relevant. Positive and negative controls performed as expected.

 

Under the conditions of this assay, diammonium hexachloropalladate displayed no significant evidence of genotoxicity in human lymphocytes, with or without metabolic activation.

Reference 7

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 October 2013 - 29 January2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Expt 1 cultures were not gently agitated during the entire 3 hour treatment incubation period; the actual duration of agitation is not known. Neither interpretation of study findings nor integrity of study are compormised by this.

GLP compliance:

yes

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):

- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES buffer, penicillin, streptomycin, amphotericin B, sodium pyruvate acid, heat-inactivated horse serum at 0, 10 or 20% and pluronic (in the 0 and 10% horse serum groups only)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

MolTox(TM) S-9

Test concentrations with justification for top dose:

In the range-finder, with and without S9:
0.3438, 0.6875, 1.375, 2.75, 5.50, 11.0 ug/ml

In experiment 1:
2, 4, 6, 8, 10, 11 ug/ml

In experiment 2:
1.5, 3, 4.5, 6, 7.5, 9, 11 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (dimethyl formamide)
- Justification for choice of solvent/vehicle: test substance solubility in DMF, not seen in other tested solvents (water, ethanol, acetone or tetrahydrofuran)

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:

benzo(a)pyrene in presence of S9; 4-nitroquinoline-N-oxide in absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in solution

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12 days

DETERMINATION OF CYTOTOXICITY
- Method: relative survival

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 control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend
analysis (p≤0.05)
3. The 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 that only partially satisfied the assessment criteria described above were
considered on a case-by-case basis.

Statistics:

All calculations were performed by computer using validated software and 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Diammonium hexachloropalladate did not induce mutations in a mouse lymphoma assay in vitro, when tested up to a limit of solubility in the presence and absence of rat liver metabolic activation.

Executive summary:

Diammonium hexachloropalladate was tested 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. It was tested in mouse lymphoma (L5178Y) cells at multiple concentrations up to the limit of solubility (11 µg/ml) in the presence and absence of rat liver (S9) metabolic activation.

There was a minor increase in mutant frequency at a single low concentration in one of the two duplicate experiments that was within the acceptable range for vehicle controls, and was not considered biologically relevant. There were no other statistically significant increases in mutations, and no linear trend was observed. In conclusion, the test material was considered non-mutagenic to mouse lymphoma cells in vitro under the conditions of this assay.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

17 October 2013 - 29 January2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Expt 1 cultures were not gently agitated during the entire 3 hour treatment incubation period; the actual duration of agitation is not known. Neither interpretation of study findings nor integrity of study are compormised by this.

GLP compliance:

yes

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):

- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES buffer, penicillin, streptomycin, amphotericin B, sodium pyruvate acid, heat-inactivated horse serum at 0, 10 or 20% and pluronic (in the 0 and 10% horse serum groups only)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

MolTox(TM) S-9

Test concentrations with justification for top dose:

In the range-finder, with and without S9:
0.3438, 0.6875, 1.375, 2.75, 5.50, 11.0 ug/ml

In experiment 1:
2, 4, 6, 8, 10, 11 ug/ml

In experiment 2:
1.5, 3, 4.5, 6, 7.5, 9, 11 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (dimethyl formamide)
- Justification for choice of solvent/vehicle: test substance solubility in DMF, not seen in other tested solvents (water, ethanol, acetone or tetrahydrofuran)

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:

benzo(a)pyrene in presence of S9; 4-nitroquinoline-N-oxide in absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in solution

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12 days

DETERMINATION OF CYTOTOXICITY
- Method: relative survival

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 control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend
analysis (p≤0.05)
3. The 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 that only partially satisfied the assessment criteria described above were
considered on a case-by-case basis.

Statistics:

All calculations were performed by computer using validated software and 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Diammonium hexachloropalladate did not induce mutations in a mouse lymphoma assay in vitro, when tested up to a limit of solubility in the presence and absence of rat liver metabolic activation.

Executive summary:

Diammonium hexachloropalladate was tested 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. It was tested in mouse lymphoma (L5178Y) cells at multiple concentrations up to the limit of solubility (11 µg/ml) in the presence and absence of rat liver (S9) metabolic activation.

There was a minor increase in mutant frequency at a single low concentration in one of the two duplicate experiments that was within the acceptable range for vehicle controls, and was not considered biologically relevant. There were no other statistically significant increases in mutations, and no linear trend was observed. In conclusion, the test material was considered non-mutagenic to mouse lymphoma cells in vitro under the conditions of this assay.

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).

Dipotassium hexachloropalladate was assessed for mutagenic activity in a bacterial reverse mutation (Ames) assay, similar to OECD Test Guideline 471, using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 and tested in the presence and absence of a rat liver metabolic activation (S9) system. (The recommended strain TA1535 was omitted.) No mutagenic effect was seen in any of the four strains at up to cytotoxic concentrations, either in the presence or absence of S9 (Bunger et al., 1996). In support, in a limited Ames test, dipotassium hexachloropalladate was not mutagenic in two strains of S. typhimurium (TA98 and TA100) when tested at up to 1 mg/plate, in the absence of metabolic activation (Suraikina et al., 1979).

  

As there are some limitations in the existing bacterial mutagenicity database for dipotassium hexachloropalladate (i.e. the recommended strain TA1535 was not tested), it was prudent to also consider the closely-related substance diammonium hexachloropalladate, which has recently been assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Test Guideline 471. Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were tested with and without the addition of rat liver S9, in two separate experiments. In the first experiment, agar containing the test substance at up to 110 µg/plate was incubated with the bacterial strains for 3 days. The second experiment, also using concentrations of up to 110 µg/plate, included an additional 20-minute pre-incubation step for cultures treated in the presence of S9. No evidence of mutagenicity was observed in any experiment. Cytotoxicity was observed from 34.79 and/or 110 µg/plate in all strains, with and without S9. In the second experiment, cytotoxicity was seen from 55 and/or 110 µg/plate. Vehicle and positive controls performed as expected. Under the conditions of this assay, diammonium hexachloropalladate was not mutagenic (Mc Garry, 2014).

No studies investigating the genotoxicity of dipotassium hexachloropalladate in mammalian systems were identified. However, diammonium hexachloropalladate was tested 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. It was tested in mouse lymphoma (L5178Y) cells at multiple concentrations up to the limit of solubility (11 µg/ml) in the presence and absence of rat liver (S9) metabolic activation. There was a minor increase in mutant frequency at a single low concentration in one of the two duplicate experiments that was within the acceptable range for vehicle controls, and was not considered biologically relevant. There were no other statistically significant increases in mutations, and no linear trend was observed. In conclusion, the test material was considered non-mutagenic to mouse lymphoma cells in vitro under the conditions of this assay (Lloyd, 2014a).

Further, diammonium hexachloropalladate has been investigated in a reliable in vitro micronucleus test, conducted to GLP and according to OECD Test Guideline 487. Following a range-finding study, whole blood was obtained from two healthy male volunteers. Blood was treated with the test substance (in dimethylformamide) at up to the limit of solubility (11 µg/ml), with or without rat liver S9. Treatment was either continuous (24 hours, without S9 only), or for 3 hours, followed by a 21-hour recovery phase (with and without S9). The only statistically significant increased frequency of micronucleated lymphocytes was seen for cultures treated with 8 µg/ml for 24 hours without S9. As other cultures at this - and higher concentrations - demonstrated no such effect, this was not considered biologically relevant. Positive and negative controls performed as expected. Under the conditions of this assay, diammonium hexachloropalladate displayed no significant evidence of genotoxicity in human lymphocytes, with or without the addition of S9 (Lloyd, 2014b).

Diammonium hexachloropalladate is considered to fall within the scope of the read-across category "hexachloropalladate salts". See section 13 for full justification report.

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 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 reproductive/developmental toxicity screening assay. As such, classification of diammonium hexachloropalladate or dipotassium hexachloropalladate for germ cell mutagenicity is not warranted, according to EU CLP criteria (EC 1272/2008).