Di(tetraethylammonium)hexahydroxoplatinate(IV)

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:

2019

Report date:

2019

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:

platinumhexol; bis(tetrapropylazanium)

EC Number:

848-537-7

Cas Number:

1912392-88-1

Molecular formula:

C16H46N2O6Pt

IUPAC Name:

platinumhexol; bis(tetrapropylazanium)

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: 3341/00-F9
- Expiration date of the lot/batch: September 10, 2020
- Purity test date: Certificate of analysis dated September 09, 2019
- Purity: 31.3 % w/w
- Form: liquid
- Appearance: amber liquid

Method

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:

Sprague-Dawley rat liver induced with phenobarbital-5,6 and benzoflavone

Test concentrations with justification for top dose:

In the plate incorporation method test the maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

Sterile distilled water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 4

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Controls 6

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent 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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the mutation test employing the plate incorporation method.
A test item precipitate (white and particulate in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in the mutation test. This observation did not prevent the scoring of revertant colonies.
In the absence of metabolic activation, statistically significant increases in TA100 revertant colony frequency were noted from 50 μg/plate, although this particular response was quite flat over the statistically significant concentrations. However, dose-related and statistically significant increases were noted for WP2uvrA from 15 μg/plate and TA98 from 1500 μg/plate. The increases observed for WP2uvrA were particularly large and in excess of the upper in-house historical control maxima range with a maximum fold increase of 7.1 fold over the concurrent vehicle control noted at 5000 μg/plate. A 2.4 fold increase over the concurrent vehicle control was noted for TA98 at 5000 μg/plate.
Larger increases were noted in the presence of metabolic activation with dose-related and statistically significant increases noted from 150 μg/plate for TA100 and TA98 and
50 μg/plate for WP2uvrA. The increases observed for each strain were large and in excess of the upper in-house historical control maxima range with maximum fold increases over the concurrent vehicle controls noted of 2.3 for TA100, 7.2 for WP2uvrA and 3.8 for TA98.
Any excursions outside the maxima ranges, particularly when a dose-response relationship are apparent, must be considered to be evidence of a biological response.
No increases in colony frequency were noted at any test item concentration for bacterial strains TA1535 and TA1537 dosed in either the absence or presence of metabolic activation (S9-mix).

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 di(tetraethylammonium)hexahydroxoplatinate 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 di(tetraethylammonium)hexahydroxoplatinate was considered to be mutagenic.

Executive summary:

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

Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Reproducible, large and dose-related increases in the number of revertant colonies, which were more than two-fold the control values, with TA98, TA100 and WP2 uvrA tester strains in the plate incorporation assays, both in the presence and absence of S9 metabolism were observed. No increases in colony frequency were noted at any test item concentration for bacterial strains TA1535 and TA1537 dosed in either the absence or presence of metabolic activation.

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 di(tetraethylammonium)hexahydroxoplatinate was mutagenic in S. typhimurium and E.coli under the reported experimental conditions.