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Toxicological information

Genetic toxicity: in vitro

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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:
24 to 9 September 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study, to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
substance was mixed with 3 ml surface agar, whereas the guideline recommends 2 ml
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): platinum (2+) tetraamimine (SP-4-1) diacetate
- Substance type: no data
- Physical state: white crystalline powder
- Analytical purity: 95.2%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: Pt 48.75%, Cl 0.029%
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: GB 379
- Expiration date of the lot/batch: 28 June 2005
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature in the dark

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9 microsomal fraction obtained from the livers of male Wistar rats
Test concentrations with justification for top dose:
In a dose-finding assay, concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg per plate were tested in triplicate on TA100 and WP2uvrA, with and without metabolic activation. Both the mutagenic and cytotoxic effects of the test material on these strains was analysed. As no cytotoxic potential was observed, subsequent mutagenicity testing on strains TA98, TA1535 and TA1537 used concentrations of 100, 333, 1000, 3330 and 5000 µg platinum(2+) tetraamimine (SP-4-1) diacetate per plate (with and without metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Applied to S. typhimurium strain TA1535, without metabolic activation, only (at 5 µg per plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Applied to S. typhimurium strain TA1537, without metabolic activation, only (at 60 µg per plate) Migrated to IUCLID6: in Milli-Q water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Applied to S. typhimurium strain TA98, without metabolic activation, only (at 10 µg per plate) Migrated to IUCLID6: in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Applied to S. typhimurium strain TA100, without metabolic activation, only (at 650 µg per plate) Migrated to IUCLID6: in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Applied to E. coli strain WP2uvrA, without metabolic activation, only (at 10 µg per plate) Migrated to IUCLID6: in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO
Remarks:
Applied to all strains (1 µg per plate for S. typhimurium strains TA98, TA100 and TA1535; 2.5 µg per plate for S. typhimurium strain TA1537; 10 µg per plate for E. coli strain WP2uvrA), with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: n/a
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): no data

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not relevant
- Determination of endoreplication: not relevant
- Other: no data
Evaluation criteria:
A test substance is considered positive (mutagenic) in the test if it induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. Any mean plate count of less than 20 is considered to be not biologically relevent. A test substance is considered to be negative (not mutagenic) if the total number of revertants in any tested strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation. These results should be reproducible in at least one independently repeated experiment.
Statistics:
No formal hypothesis testing was performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
platinum (2+) tetraamimine (SP-4-1) diacetate induced up to a 13-fold dose-related increase in the number of revertant colonies compared to the solvent control
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
platinum (2+) tetraamimine (SP-4-1) diacetate induced up to a 18-fold dose-related increase in the number of revertant colonies compared to the solvent control
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
platinum (2+) tetraamimine (SP-4-1) diacetate induced up to a 2.3-fold dose-related increase in the number of revertant colonies compared to the solvent control
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
platinum (2+) tetraamimine (SP-4-1) diacetate induced up to a 3.2-fold dose-related increase in the number of revertant colonies compared to the solvent control
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
platinum (2+) tetraamimine (SP-4-1) diacetate induced up to a 6-fold dose-related increase in the number of revertant colonies compared to the solvent control
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA100 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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: platinum (2+) tetraamimine (SP-4-1) diacetate did not precipitate in top agar, or on plates at the start and end of the incubation period

RANGE-FINDING/SCREENING STUDIES: platinum (2+) tetraamimine (SP-4-1) diacetate caused no cytotoxicity in S. typhimurium strain TA100 or E. coli strain WP2uvrA when tested at up to 5 mg per plate

COMPARISON WITH HISTORICAL CONTROL DATA: laboratory background historical ranges were presented for negative (number of spontaneous revertants per plate) and positive control data for each of the tested strains. Experimental control results were compared to these values.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Applicant's summary and conclusion

Conclusions:
In a GLP study performed according to OECD Test Guideline 471, tetraammineplatinum diacetate was mutagenic in Salmonella typhimurium strains TA98 and TA1537 and Escherichia coli strain WP2uvrA when tested aat up to 5 mg/plate in the presence and absence of metabolic activation.
Executive summary:

The genotoxic potential of tetraammineplatinum diacetate was analysed in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP. A dose range finding test was performed using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2uvrA. Triplicate cell cultures were exposed to tetraammineplatinum diacetate at up to 5000 µg/plate, both in the presence and absence of metabolic activation by rat liver fraction S9 (alongside appropriate vehicle and positive controls). These cultures were then inspected for signs of cytotoxicity and for the presence of revertant colonies. Since no cytotoxicity was observed, in the main experiment triplicate cultures of S. typhimurium strains TA98, TA1535 and TA1537 were exposed to the test material at concentrations of 100, 330, 1000, 3330 and 5000 µg/plate, both in the presence and absence of metabolic activation by S9 (alongside appropriate vehicle and positive controls), and were incubated for 48 hours at 37 °C before being inspected for signs of cytotoxicity and for the presence of revertant colonies.

Significant cytotoxicity was not observed for any of the tested strains, at any of the tested concentrations. Significant, dose-related increases in the number of observed revertant colonies were seen for S. typhimurium strains TA98 (up to 2.3 -fold in the presence of S9) and TA1537 (up to 13 -fold and 18 -fold in the absence and presence of S9 respectively), and E. coli strain WP2uvrA (up to 3.2 -fold and 6 -fold in the absence and presence of S9 respectively). No significant, dose-related increases were seen in S. typhimurium strain TA98 in the absence of S9, or in TA100 and TA1535 (both in the absence and presence of S9). Under the conditions of this assay, tetraammineplatinum diacetate showed mutagenic potential.