Diammonium hexachloropalladate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August to 30 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Good-quality guideline study, performed to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

19168-23-1

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): diammonium hexachloropalladate
- Substance type: no data
- Physical state: brown-reddish crystalline solid
- Analytical purity: ~100%
- Impurities (identity and concentrations) (ppm with respect to metal): gold (11), calcium (4), iron (1), potassium (45), sodium (40), rhodium (1)
- Composition of test material, percentage of components: 30.09% palladium
- Isomers composition: no data
- Purity test date: 9 July 2013
- Lot/batch No.: DZ0192
- Expiration date of the lot/batch: 7 July 2018
- Stability under test conditions: no data
- Storage condition of test material: tightly sealed container at 15-25°C, protected from light

Method

Target gene:

Histidine locus

Species / strain

Species / strain 1

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 2

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)

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In a good-quality Ames assay, conducted to GLP and OECD 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 system.

Executive summary:

Diammonium hexachloropalladate has been assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD 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 to Salmonella at up to 110 µg/plate, with and without S9