Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2007 to 26 June 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study, to GLP
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 November 2007 to 5 December 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Cells tested only in the presence of metabolic activation
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
historical control data not provided, cells only treated in the presence of metabolic activation (this confirmatory study should be considered alongside Krebs et al. (2007), which contains sufficient data for cells treated in the absence of S9)
Qualifier:
according to guideline
Guideline:
other: EEC Council Directive 2000/32, Annex 4A (adapting 67/548/EWG Annex V/part B.10)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F.10 (499 ml), Streptomycin sulphate 50 mg/ml & Penicillin G 50,000 IU/ml (1 ml), newborn calf serum (88.2 ml)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: the report states that "the karyotype, generation time and plating efficiency have been checked in this laboratory"
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with
Metabolic activation system:
Phenobarbital and betanaphthoflavone-induced rat liver tissue fraction S9
Test concentrations with justification for top dose:
156, 313, 625, 1250, 2500 and 5000 µg/ml [equivalent to 31.2, 62.6, 125, 250, 500 and 1000 µg/plate]
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: at 15 and 23 µg/ml
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
As part of a guideline study, conducted to GLP, tetraammineplatinum diacetate did not induce chromosome aberrations in Chinese hamster ovary cells in vitro, in the presence of metabolic activation.
Executive summary:

The clastogenic potential of tetraammineplatinum diacetate was confirmed in a subsequently conducted in vitro study on Chinese hamster ovary cells.

 

Duplicate cultures were treated with the test substance at 156, 313, 625, 1250, 2500 or 5000 µg/ml [equivalent to up to 1000 µg/plate] in the presence of metabolic activation by rat liver fraction S9 (alongside appropriate vehicle, untreated, and positive controls). Following three hours of treatment, cells were harvested for twenty hours (at 37 °C).

 

Following fixation and Giemsa staining, no remarkable cytotoxicity was observed over the whole dose-range, so cultures treated with the three highest tested concentrations (1250, 2500 and 5000 µg/ml) were scored for chromosome aberrations.

 

No significant treatment-related increase was observed in the number of cells displaying chromosomes with gaps, deletions or exchanges, or in the number of polyploid or endoreplicated cells. It was therefore determined that tetraammineplatinum diacetate was not clastogenic in Chinese hamster ovary cells in vitro, in the presence of metabolic activation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
historical control data not provided
Qualifier:
according to guideline
Guideline:
other: EEC Council Directive 2000/32, Annex 4A (adapting 67/548/EWG Annex V/part B10)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
127733-97-5
Cas Number:
127733-97-5
IUPAC Name:
127733-97-5
Details on test material:
- Name of test material (as cited in study report): platinum(2+), tetraammine-,(SP-4-1)-, diacetate
- Substance type: no data
- Physical state: white crystalline powder
- Analytical purity: 46.95% Pt
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 20 March 2006
- Lot/batch No.: AP013/06
- Expiration date of the lot/batch: 30 March 2007 (20 March 2007 on the label)
- Stability under test conditions: no data
- Storage condition of test material: room temperature, protected from light

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F.10 (499 ml), Penicillin G 50,000 IU/ml & streptomycin sulphate 50 mg/ml (1 ml), newborn calf serum (88.2 ml)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: the report states that "the karyotype, generation time and plating efficiency have been checked in this laboratory
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and betanaphthoflavone-induced rat liver tissue fraction S9
Test concentrations with justification for top dose:
39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/ml [equivalent to roughly 7.8, 15.6, 31.2, 62.6, 125, 250, 500 and 1000 µg/plate]
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (sterile, distilled)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water (10%)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: at 0.3 and 0.45 µg/ml, without S9
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water (10%)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: at 15 and 23 µg/ml, with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no data
- Exposure duration: 3 hours (assay one); 20 hours (continuous treatment to sampling, assay two)
- Expression time (cells in growth medium): 20 hours (assay one)
- Fixation time (start of exposure up to fixation or harvest of cells): 23 hours (assay one); 20 hours (assay two)

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 100 per culture, 50 per culture in positive controls due to high incidence of aberrant cells (at least 50%)

DETERMINATION OF CYTOTOXICITY
- Method: relative cell growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The test item was determined to be clastogenic if statistically significant increases were seen in the incidence of cells bearing aberrations at any dose-level over the concurrent control, exceeding historical control values, and reproduced in both replicate cultures.
Statistics:
Fisher's Exact Test was used to compare the number of cells bearing aberrations in control and treated cultures.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cell count slightly reduced (86% compared to control) at highest tested concentration (5000 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cell count slightly reduced (85% compared to control) at highest tested concentration (5000 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH monitored
- Effects of osmolality: osmolarity monitored
- Evaporation from medium: no data
- Water solubility: test compound was soluble in water at 5000 µg/ml
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: not provided

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data
Remarks on result:
other: other: assay one (3-hour treatment)
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
In a guideline study, to GLP, tetraammineplatinum(II) diacetate did not induce chromosome aberrations in Chinese hamster ovary cells in vitro, both in the absence and presence of metabolic activation.
Executive summary:

The clastogenicity of tetraammineplatinum(II) diacetate was assessed in an in vitro study on Chinese hamster ovary cells, conducted according to OECD Test Guideline 473 and to GLP.

 

Duplicate cultures were treated with the test substance at 39.1, 78.1, 156, 313, 625, 1250, 2500 or 5000 µg/ml [equivalent to up to 1000 µg/plate], both in the presence and absence of metabolic activation by rat liver fraction S9 (alongside appropriate vehicle, untreated, and positive controls). Following three hours of treatment, cells were harvested for twenty hours (at 37 °C). In a second assay, cells were treated with the test substance continuously until sampling at 20 hours, in the absence of metabolic activation.

 

Cells were checked for cytotoxicity following fixation and Giemsa staining. The cell count was slightly reduced to 85% of the control in cultures treated with 5000 µg/ml in the presence of S9. This level of cytotoxicity did not impede metaphase spread analysis, so cultures treated with the three highest tested concentrations (1250, 2500 and 5000 µg/ml) were scored for chromosome aberrations. In the second assay, the cell count was reduced to 86% of the control at the highest dose, but no cytotoxicity was observed over the remaining dose-range. In this case, cultures treated with 625, 1250 and 2500 µg/ml were selected for metaphase spread analysis.

 

No significant treatment-related increase was observed in the number of cells displaying chromosomes with gaps, deletions or exchanges, or in the number of polyploid or endoreplicated cells. It was therefore determined that tetraammineplatinum diacetate was not clastogenic in Chinese hamster ovary cells in vitro.