[No public or meaningful name is available]

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphato, nitrito, sulfato, amino, aquo platinate (II) complex

IUPAC Name:

Phosphato, nitrito, sulfato, amino, aquo platinate (II) complex

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 4133/00-K0
- Expiration date of the lot/batch: 07 June 2021
- Purity test date: Certificate of analysis dated 04 December 2020
- Form: liquid
- Appearance: Clear yellow liquid

Method

Target gene:

Histidine for S. typhimurium strains; tryptophan for E.coli WP2 uvrA

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
The S9 batch is characterised with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene (Sigma), which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assay was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The mutation experiment was a direct plate assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Vehicle / solvent:

Sterile distilled water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Controls 2

Positive control substance:

sodium azide

Remarks:

5 µg/plate for TA1535 without S9

Controls 3

Positive control substance:

other:

Remarks:

2.5 µg/plate for TA1537 without S9

Controls 4

Positive control substance:

2-nitrofluorene

Remarks:

10 µg/plate for TA1537 and TA98 without S9

Controls 5

Positive control substance:

methylmethanesulfonate

Remarks:

650 µg/plate for TA100 without S9

Controls 6

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

10 µg/plate for WP2uvrA without S9

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 µg/plate for TA1535 and TA1537, 1 µg/plate for TA98 and TA100, 15 µg/plate for WP2uvrA with S9

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
• The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and unless the total number of revertants in tester strain TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
A test item is considered positive (mutagenic) in the test if:
• The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three times the concurrent control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation Experiment: Direct Plate Assay
The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and
5000 μg/plate.
Precipitate
Precipitation of the test item on the plates was not observed in any tester strain.
Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.
Mutagenicity
In tester strain TA100, both in the absence and presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 5.1- fold the concurrent control. The increases were outside the historical control data range and the increases were more than two-fold the concurrent control.
In tester strain TA98, both in the absence and presence of S9-mix, the test item induced
dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 28 and 19- fold the concurrent control, respectively. The increases were outside the historical control data range and more than three-fold the concurrent control.
In tester strain WP2uvrA, both in the absence and presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 2.4 and 2.3- fold the concurrent control, respectively. The increases were within the historical control data range but more than two-fold the concurrent control.

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Pt concentrate P induced substantial increases in the frequency of TA100, WP2uvrA and TA98 revertant colonies both with and without metabolic activation (S9-mix). Under the conditions of this test Pt concentrate P was considered to be mutagenic.

Executive summary:

In an OECD Test Guideline 471 study, conducted according to GLP, Pt concentrate P was assessed for its ability to induce gene mutations in strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2 uvrA).

The test item induced dose-related and biologically relevant increases in the number of revertant colonies compared to the solvent control in tester strain TA100, TA98 and WP₂uvrA.

No second experiment, including a pre-incubation step, was performed as the OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment.

 

It was concluded that Pt concentrate P was mutagenic in S. typhimurium and E.coli under the reported experimental conditions.