Tetraammineplatinum dichloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tetraammineplatinum dichloride displayed evidence of genotoxicity in several published in vitro gene mutation assays with bacterial cells (Bootman and Lodge, 1980a; Suraikina et al., 1979; Uno and Morita, 1993). In mammalian cells, the substance did not induce gene mutations in an early study (Johnson et al., 1980), though displayed evidence of mutagenicity in a more recent investigation (Lloyd, 2017); the structurally-related compound, tetraammineplatinum hydrogen carbonate, was also considered to be mutagenic (Durward, 1998a, b). Another related compound, tetrammineplatinum diacetate, did not induce chromosome aberrations in a mammalian cell line (Ciliutti et al., 2007; 2008) but was positive in an in vitro gene mutation assay with bacterial cells (Verspeek-Rip 2004).

Link to relevant study records

Reference

Reference 1

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD guidelines, with certain deviations.

Remarks:

Lacks a strain capable of detecting cross-linking mutagens, only tested at up to 1 mg/plate (instead of the recommended 5 mg/plate) and only plated in duplicate rather than in triplicate.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Guideline recommends the use of a strain capable of detecting cross-linking mutagens, which was not done. Only tested at up to 1 mg/plate (instead of the recommended 5 mg/plate). Only plated in duplicate rather than in triplicate.

Principles of method if other than guideline:

Method carried out according to Ames et al. (1975). Mutation Research 31, 347-364.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Metabolic activation system:

Rat liver microsomal fraction (S9) from male, CD1 rats induced with Aroclor 1254

Test concentrations with justification for top dose:

1.6, 8.0, 40, 200 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water (uncoded batch); isotonic saline (batch 031099/A)
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N- nitrosoguanidine

Remarks:

10 µg/plate for TA1535 and TA100 without S9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

20 µg/plate for TA1537, TA 98 and TA100, with and without S9

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate for TA1537 without S9

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

20 µg/plate for TA1538 and TA98 without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hr

OTHER: Plates prepared in duplicate.
Only tested up to 1 mg/ml although no problem with solubility was reported.
Tests with TA1537, TA98 and TA100 were conducted twice, both with and without S9 using the “uncoded” test compound in water. Three separate tests were performed with batch 031099/A dissolved in isotonic saline and TA1535, TA1537 and TA1538, only without S9.

Evaluation criteria:

No data, but reported the method to be according to Ames et al. (Mutation Research 1975, 31, 347-364), who considered a less than 2-fold increase in revertants, compared to spontaneous revertants, to be a negative response.

Statistics:

No data

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: weak positive

Cytotoxicity / choice of top concentrations:

other: cytotoxic at 1000 μg/plate in one of the duplicate tests, both in the presence and absence of S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

other: weak positive

Cytotoxicity / choice of top concentrations:

other: cytotoxic at 1000 μg/plate in one of the duplicate tests, both in the presence and absence of S9

Vehicle controls validity:

valid

Positive controls validity:

valid

TEST-SPECIFIC CONFOUNDING FACTORS No data   RANGE-FINDING/SCREENING STUDIES: no data   COMPARISON WITH HISTORICAL CONTROL DATA: no data in the report [but revertants within the ranges given in the literature].   ADDITIONAL INFORMATION ON CYTOTOXICITY: in one of the duplicate tests there was a thinning of the background lawn reported for TA98 and TA100 together with a decrease in the number of revertants, both with and without S9. This was not evident with TA1537, or for TA 1535 and TA 1538 which were only tested without S9.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

In a limited study, tetraammineplatinum dichloride showed a dose-related mutagenic activity in TA 1537, and a weak positive response (about a 2-fold increase in mutant frequency, compared to the spontaneous frequency) in TA98 and TA100, both in the presence and absence of metabolic activation.

Executive summary:

Tetraammineplatinum dichloride was tested for mutagenic activity by reversion to histidine independence in five strains of Salmonella typhimurium, TA1535, TA1537, TA 1538, TA98 and TA100, using pour-plate assays.

 

Duplicate assays were carried out with strains TA98, TA100 and TA1537, both with and without a rat liver microsomal fraction (S9), using an aqueous solution of the test item. Three separate tests were conducted with strains TA1535, TA1537 and TA1538 in the absence of S-9 mix only, using the test substance dissolved in isotonic saline solution. In all experiments, test doses ranged from 1.6 to 1000 µg/plate (lower than the recommended top concentration).

 

A dose-related increase in mutant frequency (13- to 36-fold above that for spontaneous mutants) was observed in TA 1537, which detects frameshift mutations. A weak positive response (about a 2-fold increase in mutant frequency, compared to the spontaneous frequency) was also observed in TA98 and TA100, which also detect frameshift mutations. These increases in mutant frequencies were seen both in the presence and absence of S9.No increase was seen with strains TA1535 and TA1538, either in the presence or absence of S9.

Reference 2

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:

Conducted according to GLP. The lack of historical control data is not anticipated to affect the outcome of the 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

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

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)

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.

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'.

Conclusions:

Interpretation of results (migrated information):
negative

In a guideline study, conducted to GLP, platinum(2+), tetraammine-,(SP-4-1)-, diacetate did not induce chromosomal aberrations in Chinese hamster ovary cells in vitro, both in the absence and presence of metabolic activation.

Executive summary:

The clastogenicity of platinum(2 +), tetraammine-,(SP-4 -1)-, 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 platinum(2+), tetraammine-,(SP-4-1)-, diacetate was not clastogenic in Chinese hamster ovary cells in vitro.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: Read-across from study conducted on a member of the "tetraammineplatinum(II) salts" category, tetraammineplatinum diacetate

Adequacy of study:

key study

Study period:

20 February 2007 to 26 June 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study, conducted to GLP, on read-across compound. The lack of historical control data is not anticipated to affect the outcome of the study.

Justification for type of information:

substance is considered to fall within the scope of the read-across category "tetraammineplatinum(II) salts".

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

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

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)

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.

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'.

Conclusions:

Interpretation of results (migrated information):
negative

In a guideline study, conducted to GLP, platinum(2+), tetraammine-,(SP-4-1)-, diacetate did not induce chromosomal aberrations in Chinese hamster ovary cells in vitro, both in the absence and presence of metabolic activation.

Executive summary:

The clastogenicity of platinum(2 +), tetraammine-,(SP-4 -1)-, 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 platinum(2+), tetraammine-,(SP-4-1)-, diacetate was not clastogenic in Chinese hamster ovary cells in vitro.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October - 22 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Thymidine kinase (tk) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Growth media (RPMI 1640; containing L-glutamine and HEPES) prepared with horse serum, penicillin, streptomycin, amphotericin B, sodium pyruvate acid and pluronic; tissue culture flasks were gassed with 5±1% v/v CO2 in air.
- Properly maintained: yes. The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells were stored as frozen stocks in liquid nitrogen.
- Periodically checked for Mycoplasma contamination: yes. Each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: yes. Each batch of frozen cells was purged of mutants.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) from male Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Cytotoxicity range-finder experiment: 54.82, 109.6, 219.3, 438.6, 877.2 and 1754 μg/mL (3-hr treatment; both with and without S9) and 7.813, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL (24-hr treatment; without 29)
Mutation experiment: 100, 200, 400, 800, 1200, 1600 and 2000 μg/mL (3-hr treatment; both with and without S9) and 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 and 750 μg/mL (24-hr treatment; without 29)

In the mutation experiment, the highest dose levels were informed by the observed effects on relative suspension growth (RSG) with increasing test concentration in the range-finder experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified water diluted 10-fold in the treatment medium
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. For 3 hour treatments in the absence and presence of S-9, at least 10^7 cells in a volume of 17 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were used. For 24 hour treatment in the absence of S-9 at least 4 x 10^6 cells in a volume of 18 mL RPMI 10 were used. For all treatments 2 mL vehicle or test article or 0.2 mL positive control solution (plus 1.8 mL of purified water) was added to the cell suspensions. For 3 hour treatments, S-9 mix or 150 mM KCl was added, as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment culture volume was 20 mL. After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 x g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 x g) for 5 minutes and resuspended in 50 mL RPMI 10 medium. The 24 hour experiment similarly involved static incubation at 37±1°C and subsequent steps (albeit in 20 mL RPMI 10 medium). Cell densities were determined using a Coulter counter and adjusted to 2 x 10^5 cells/mL. Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment.


DURATION
- Preincubation period: Not applicable
- Exposure duration: 3 hours (with and without S9); 24 hours (without S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: For viability (192 wells averaging 1.6 cells/well) and for TFT resistance (384 wells at 2000 cells/well).

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test substance was measured by calculating the relative total growth (RTG) according to the following formula: RTG = RSG x ([Individual viability value]/[Mean control viability value]), where RSG = ([Individual SG value]/[Mean control SG value]) x 100. Viability (cloning efficiency [CE]) was calculated as follows: CE = ([-ln P(0)]/[Number of cells per well]) x 100, where P(0) = (Number of wells with no colony)/(Total number of wells)

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the vehicle control mutant frequency plus GEF
2. The linear trend test was statistically significant.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Linear regression was performed on ranked mutant frequency against ranked concentration to test for a linear trend. The linear trend was performed on the overall MF per concentration level, not individual replicates (i.e. one MF value per concentration). The test for linear trend is one-tailed, therefore negative trend was not considered relevant. The positive control was excluded from the test for linear trend.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

3-hr: MF increases exceeding the GEF were observed at 400-2000 (-S9) and 200-2000 (+S9) µg/mL 24-hr: MF increases exceeding the GEF were observed at 100-450 µg/mL (-S9) Statistically significant linear trends were observed in all 3 experiments.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest concentrations analysed in the 3-hr treatment experiment (+ and - S9; both 2000 μg/mL) gave 11 and 45% RTG, respectively. The highest concentrations analysed in the 24-hr treatment experiment (- S9; 450 μg/mL) gave 11% RTG.

Vehicle controls validity:

valid

Remarks:

MF in vehicle control cultures fell within acceptable ranges.

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Clear increases in mutation were induced by the positive control chemicals.

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No marked changes in osmolality or pH were observed in the 3 and 24 hour Range Finder Experiment at the highest concentration tested (2000 µg/mL), compared to the concurrent vehicle controls
- Evaporation from medium: No data
- Water solubility: Preliminary solubility data indicated that Tetraammine platinum dichloride was soluble in purified water at a concentration of at least 25.76 mg/mL.
- Precipitation: The solubility limit in culture medium was at least 2576 µg/mL, as indicated by the absence of precipitate at this concentration approximately 24 hours following test article addition.
- Definition of acceptable cells for analysis: In the 3-hr experiment, all concentrations were selected to determine viability and TFT resistance. In the 24-hr experiment, the highest concentration tested in the absence of S-9 (750 µg/mL) was considered too toxic for selection to determine viability and TFT resistance. In addition an intermediate concentration (400 µg/mL) was not selected as it was considered unrepresentative of the remainder of the data set. All other concentrations were selected, aside from two (500 and 600 µg/mL) which were rejected due to extreme toxicity.

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment (3 hour treatment) six concentrations were tested, in the absence and presence of S-9, ranging from 62.5 to 1754 µg/mL, which was broadly in accordance with the recommendations of the regulatory test guidelines. No post treatment precipitate was observed. The highest concentration tested, 1754 µg/mL, gave 51% and 37% RSG in the absence and presence of S-9 at the end of the expression period. Accordingly, the maximum concentration analysed in the Mutation Experiment was 2000 µg/mL, in compliance with the regulatory test guidelines.

In the cytotoxicity Range-Finder Experiment (24 hour treatment) nine concentrations were tested in the absence of S-9, ranging from 7.813 to 2000 µg/mL. The highest concentration to give ≥10% RSG at the end of the expression period was 250 µg/mL, which gave 40% RSG.


MUTAGENICITY (INCLUDING COMPARISON WITH HISTORICAL CONTROL DATA):
In the 3-hr experiment, increases in MF exceeding the GEF (126 mutants per 10^6 viable cells) were observed at the highest five concentrations in the absence of S-9 (400-2000 µg/mL) and at the highest six concentrations in the presence of S-9 (200-2000 µg/mL). Statistically significant linear trends (p≤ 0.01) were observed under both treatment conditions.

In the 24-hr experiment (in the absence of S-9), MF increases exceeding the GEF were observed at the highest seven concentrations (100-450 µg/mL); there was a statistically significant linear trend (p≤ 0.05).

Under all three treatment conditions the data were indicative of a clear positive result.

For the vehicle controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 27% to 35% in the Mutation Experiment. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals (MMS and B[a]P). At the test article concentrations which exceeded the GEF, increases in both small and large colony MF were observed and there were clear increases in the proportions of small colony mutants under each treatment condition.

Under all three treatment conditions the data were indicative of a clear positive result.

No comparison with historical control data was made.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The highest concentrations analysed in the 3-hr treatment experiment with and without S9 (2000 μg/mL) gave 11 and 45% RTG, respectively. The highest concentration analysed in the 24-hr treatment experiment without S9 (450 μg/mL) gave 11% RTG.

The MF in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals. As such, the study was acepted as valid.

Conclusions:

In a guideline in vitro mammalian cell gene mutation assay, to GLP, tetraammine platinum dichloride induced mutations at the tk locus of L5178Y mouse lymphoma cells when tested at up to cytotoxic concentrations for 3 hours in the absence and presence of S9 and for 24 hours in the absence of S9.

Executive summary:

In an in vitro mammalian cell gene mutation test using the thymidine kinase gene, conducted according to OECD Test Guideline 490 and to GLP, tetraammineplatinum dichloride (formulated in purified water) was tested for its ability to induce gene mutations at the tk locus (5-trifluorothymidine resistance [TFT]) in L5178Y mouse lymphoma cells. The study consisted of a cytotoxicity range-finder experiment followed by a mutation experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9).

 

In the range-finding study (3-hour treatment), six concentrations (54.82-1754 μg/mL) were tested (with and without S9); respective relative suspension growth (RSG) values of 51 and 37% were seen at the highest concentration in the absence and presence of S9 at the end of the expression period. An analogous range-finding study involved 24-hour treatment using nine concentrations (7.813-2000 μg/mL) in the absence of S9. The highest concentration to give ≥10% RSG at the end of the expression period was 250 µg/mL (40%).

 

In the mutation experiment, cells were exposed to test material for 3 hours at 100-2000 μg/mL in both the absence and presence of S9. The highest concentration analysed, 2000 µg/mL, gave 45% and 11% relative total growth (RTG) in the absence and presence of S9, respectively. Similarly, a 24-hour treatment experiment utilised dose levels of 50-750 μg/mL in the absence of S9. The highest concentration analysed was 450 µg/mL (RTG 11%). Appropriate vehicle and positive control treatments were included in the experiment in the absence and presence of S9.

 

In the 3-hour treatment experiment, increases in MF which exceeded the Global Evaluation Factor (GEF) were observed at 400-2000 µg/mL in the absence of S9 and at 200-2000 µg/mL in the presence of S9. When tested up to toxic concentrations for 24 hours in the absence of S9, increases in MF which exceeded the GEF were observed at 100-450 µg/mL. Statistically significant linear trends were observed under all treatment conditions. The study was accepted as valid on the basis that MF in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals.

 

Overall, tetraammineplatinum dichloride induced mutations at the tk locus of L5178Y mouse lymphoma cells, when tested up for 3 hours in the absence and presence of S9 and for 24 hours in the absence of S9.

Reference 5

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 August 2004 to 9 September 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted according to GLP

Qualifier:

according to guideline

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

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

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.

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

Conclusions:

Interpretation of results (migrated information):
positive

In an OECD Test Guideline 471 study, to GLP, platinum (2+) tetraammine (SP-4-1) diacetate was mutagenic in Salmonella typhimurium strains TA98 and TA1537 and Escherichia coli strain WP2uvrA when tested at up to 5 mg/plate in the presence and absence of metabolic activation.

Executive summary:

The genotoxic potential of platinum (2+) tetraammine (SP-4-1) 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 platinum (2 +) tetraammine (SP-4 -1) 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, platinum (2+) tetraammine (SP-4-1) diacetate showed mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genotoxicity of tetraammineplatinum dichloride, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes and to cause DNA damage, was assessed in a study following OECD 474 and 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 250, 500 or 1000 mg/kg bw/day of the test item on three consecutive days. Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes. There was no increase in % tail intensity in the liver, glandular stomach or duodenum. 

There was a statistically significant and dose-related increase (p < 0.001) in DNA damage seen in the analysis of the kidney tissue. The tail intensity in animals dosed with 500 mg/kg bw/day was 14.56%, and in animals receiving 1000 mg/kg bw/day was 12.59%. However, these tail intensity values fell within the 95% confidence limits of the historical control data (upper limit 25.55%). Histopathological examination of the tissues did not reveal evidence of toxicity. As such, this finding was considered to be equivocal evidence of a genotoxic effect (Eurlings, 2020).

In order to assess the potential to induce gene mutations in vivo, a transgenic rodent (TGR) gene mutation assay (according to OECD Test Guideline 488) is proposed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 2020 - 30 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: 9005305492.
- Expiration date of the lot/batch: 07 January 2021.
- Purity test date: CoA issued 26 November 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2 - 8 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.
- Final preparation of a solid: Test item was suspended in corn oil.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Suspension.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 138 ± 8.4 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.

IN-LIFE DATES:
From: Not specified.
To: 09 Apr 2020.

Route of administration:

oral: gavage

Vehicle:

-Vehicle(s)/solvent(s) used: corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands)
- Justification for choice of solvent/vehicle: corn oil is a widely used standard vehicle for in vivo animal experiments.
- Concentration of test material in vehicle: analytical verification confirmed that the measured test item concentrations in vehicle were 100, 104 and 106% of the nominal values for group 2, group 3 and group 4 (i.e. 250, 500 and 1000 mg/kg(bw)/d), respectively. Accuracy and homogeneity (coefficient of variation
≤ 10%) of the test item in vehicle was confirmed. "
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 mL/kg body weight
- Stability of test item in vehicle: Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10% of the initial mean sample concentration results. The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours (which exceeded the time between preparation and use of formulations).

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Daily.

Post exposure period:

Tissue samples taken 3 - 4 hours after administration of final dose.

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Maximum tolerable dose. In a preliminary dose-range finding study, 12 animals (group 1: 1 male and 1 female, group 2: 3 males and 3 females, Group 3: 1 male and 3 females) were dosed via oral gavage with 2000, 1000 and 1500 mg/kg body weight (groups 1, 2 and 3, respectively). Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg/day. These doses were therefore considered to be higher than the MTD. A dose of 1000 mg/kg/day was assumed to be the MTD.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide.
- Route of administration: Gavage.
- Doses / concentrations: A single dose of 19 mg/kg bw, dissolved in physiological saline.

Tissues and cell types examined:

Bone marrow from the femur.

Details of tissue and slide preparation:

The femurs were flushed with foetal calf serum and the cell suspension centrifuged. The supernatant was removed and a drop of the remaining cell suspension was spread across a clean slide and fixed with methanol. The slides were automatically stained with Giemsa using the Wright Stain Procedure.

Evaluation criteria:

A test item is considered positive in the micronucleus test if all of the following criteria are
met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p <
0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared
with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.


A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in the frequency of micronucleated polychromatic erythrocytes compared with
the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

The incidence of micronuclei was assessed in at least 4000 polychromatic erythrocytes per animal.


A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable for addition to the
laboratory historical control database
b) The concurrent positive controls should induce responses that are compatible with those
generated in the historical positive control database and produce a statistically significant
increase compared with the concurrent negative control. The positive control data was
analyzed by the Welch t test (inhomogeneous variances, one-sided, p < 0.05).
c) The appropriate number of doses and cells has been analysed.
d) The criteria for the selection of the highest dose are consistent with those described in the
OECD 474 guideline

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical
analysis of the micronucleus test data.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Confirmation of exposure:
Platinum was quantifiable in plasma samples from high-dose (1000 mg/kg/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3-4 hours after the third dose. Therefore it was confirmed that the animals were exposed to the test item. No test item was detected in the animals dosed with vehicle.
No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed. A slight increase was seen in all treatment groups that was within the 95% limits of the historical control data.

Treated animals showed no decrease in the PCE:NCE ratio, indicating a lack of toxicity to the bone marrow.

Characterisation data indicating whether micronuclei contain whole or fragmented chromosomes have not been generated since the in vivo MN assay was clearly negative. Moreover, this characterisation is no requirement in the OECD474 guidance.

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes
group	treatment	Dose (mg/kg body weight)	animal number	Number of micronucleated polychromatic erythrocytes (number per animal)	Number of micronucleated polychromatic erythrocytes (mean +/- SD) (1,2)		ratio polychromatic/ normochromatic erythrocytes (mean +/- SD) (1,3)
1	vehicle control	0	1	0	2,0	± 1,6	1,24	± 0,14
			2	1
			3	3
			4	4
			5	2
2	test item	25	6	3	2,2	± 0,8	1,17	± 0,23
			7	2
			8	3
			9	1
			10	2
3	test item	50	11	4	2,6	± 1,9	1,16	± 0,19
			12	0
			13	2
			14	5
			15	2
4	test item	100	16	2	2,6	± 0,9	1,06	± 0,28
			17	2
			18	3
			19	4
			20	2
6	Cyclophosphamide	19	26	38	66,8	± 36,1 (4)	0,53	± 0,30
			27	31
			28	74
			29	69
			30	122
Legend	(1) Five animals per treatment group.
	(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
	(3) The ratio was determined from at least the first 1000 erythrocytes counted.
	(4) Significantly different from corresponding control group (Welch t test, P < 0.001).

 

Dose-response relationship & statistics

Micronucleus test (Evaluation of  4000 cells)

Test item: Comparison with the corresponding vehicle control group by using the Dunnett’s test, no significant differences

positive control: p-value (one sided) <0.001, significantly different from the corresponding vehicle control group by using the Welsh t-test

 

Distribution historical  control data from experiments performed between November 2016 and November 2019.
		negative control data	positive control data
	mean number of micronucleated cells per 4000 cells	3.9	45.2
	Standard deviation	1.0	31.8
	number of observations	29	29
	lower control limit (95% control limits)	2*	-17
	upper control limit (95% control limits)	6	108
	legend: * Rounded value; unrounded value is 1.895

Conclusions:

Tetraammineplatinum dichloride did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 1000 mg/kg bw/day by gavage on three consecutive days.

Executive summary:

The in vivo clastogenicity of tetraammineplatinum dichloride, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes, was assessed in a study following OECD 474 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 250, 500 or 1000 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received a single dose of cyclophosphamide. Bone marrow was harvested from the femurs and assessed for micronuclei.

There was a slight but not statistically significant increase in micronucleated polychromatic erythrocytes in all treatment groups, but the incidences fell within the 95% limits of the historical control data. On that basis, tetraammineplatinum dichloride was concluded to be non-genotoxic under the conditions of this assay.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 2020 - 30 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016.

Deviations:

yes

Remarks:

Only 3 or 4 animals were used for the isolation of the stomach in the control, low- and high-dose groups due to a technical error. All other values were within the historical controls and the results were clearly negative, so this did not impact the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material:
9005305492.
- Expiration date of the lot/batch: 07 January 2021.
- Purity test date: CoA issued 26 November 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
In refrigerator (2 - 8 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
None.
- Final preparation of a solid: Test item was suspended in corn oil.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspension.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 138 ± 8.4 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.

IN-LIFE DATES:
From: Not specified.
To: 09 Apr 2020.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil.
- Justification for choice of solvent/vehicle: corn oil is a widely used standard vehicle for in vivo animal experiments.
- Concentration of test material in vehicle: 27.1, 54.1 or 108 mg/g (corresponding to nominal concentrations of 25, 50 and 100 mg/mL)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Stability of test item in vehicle: homogeneity of test item suspended in vehicle demonstrated for 4 hours (sufficient for the dosing of all test animals), after which unused test item formulations were discarded.

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Daily.

Post exposure period:

Tissue samples taken 3 - 4 hours after administration of final dose.

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Maximum tolerable dose. Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg bw/day in a preliminary dose range finding study.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulphonate.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw, dissolved in physiological saline, administered twice.

Tissues and cell types examined:

Cells were isolated from the liver, glandular stomach, duodenum and kidney.

Details of tissue and slide preparation:

Minced liver or kidney tissue was added to collagenase and dissolved in HBSS (saline). This suspension was shaken and centrifuged. The cell pellet was resuspended in HBSS and kept on ice prior to preparation of the slides.

Tissue from the glandular stomach and duodenum was stored on ice in "mincing buffer incomplete" (HBSS + EDTA). The surface epithelium of both the glandular stomach and duodenum was discarded as it contains a high proportion of apoptotic cells which distort the comet analysis. The cells, suspended in the buffer, were filtered though a 100 µm cell strainer and stored on ice prior to preparation of the slides.

Low melting point agarose was added to the cell suspensions and layered on a comet slide, which was then incubated for 10 - 35 minutes in the refrigerator.

Slides were kept overnight in the refrigerator, immersed in pre-chilled lysis solution. After rinsing, the slides were placed in freshly-prepared alkaline solution; electrophoresis was performed for 20 minutes (stomach and duodenum) or 30 minutes (liver and kidney). Following another rinse, the slides were immersed in absolute ethanol and allowed to dry, before staining with SYBR Gold fluorescent dye.

Evaluation criteria:

A test item was considered positive if all of the following criteria were met:
a) at least one treatment group demonstrated a statistically significant increase in % tail intensity vs. control.
b) the increase was dose-related.
c) any of the results were outside the 95% confidence limits of the historical control data.

If none of the above criteria were met, the test item was considered negative. If the data precluded making a conclusion of clearly positive or negative, the result was concluded as equivocal.

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

Kidney: Statistically significant and dose-related (p < 0.001 for the trend) increase in tail intensity, but the mean % tail intensity within the 95% limits of the historical control data. See tables 1 and 5 below

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Liver: no statistically significant increase in % tail intensity. See table 2 below

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach: no statistically significant increase in % tail intensity. See table 3 below

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Duodenum: no statistically significant increase in % tail intensity. See table 4 below

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

See tables 5 (negative control) and 6 (positive control), below, for historical control data.

Platinum was quantifiable in plasma samples from high-dose (1000 mg/kg/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3-4 hours after the third dose. Therefore it was confirmed that the animals were exposed to the test item. No test item was detected in the animals dosed with vehicle.

Table 1: Comet results (% tail intensity) for kidney.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	3.52%	± 0.72%
250 mg/kg bw/day	7.56%	± 3.00%
500 mg/kg bw/day	14.56%	± 4.58%
1000 mg/kg bw/day	12.59%	± 6.09%
EMS (positive control)	79.86%	± 4.58%

 

Table 2: Comet results (% tail intensity) for liver.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	2.23%	± 0.43%
250 mg/kg bw/day	1.80%	± 0.44%
500 mg/kg bw/day	1.73%	± 0.25%
1000 mg/kg bw/day	1.80%	± 0.52%
EMS (positive control)	81.47%	± 1.67%

 

Table 3: Comet results (% tail intensity) for glandular stomach.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	3.14%	± 0.85%
250 mg/kg bw/day	4.44%	± 1.39%
500 mg/kg bw/day	4.37%	± 0.90%
1000 mg/kg bw/day	4.15%	± 0.35%
EMS (positive control)	52.70%	± 7.21%

 

Table 4: Comet results (% tail intensity) for duodenum.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	2.18%	± 0.28%
250 mg/kg bw/day	2.63%	± 0.85%
500 mg/kg bw/day	2.86%	± 0.93%
1000 mg/kg bw/day	2.47%	± 0.51%
EMS (positive control)	34.29%	± 4.51%

 

Table 5: Historical data Comet assay Negative control

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Stomach Tail Intensity (%) Males and Females	Kidney Tail Intensity (%) Males and Females
Mean	1.96	3.06	2.45	12.10
SD	0.92	1.52	1.39	8.46
n	85	45	60	30
Lower control limit (95% control limits)	0.27	-0.86	-1.07	-1.35
Upper control limit (95% control limits)	3.65	6.97	5.96	25.55

SD = Standard deviation

n = Number of observations

Kidney: Historical control data from experiments performed in Feb 2012 – July 2019

Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019

 

Table 6: Historical data Comet assay Positive control (200 mg/kg bw EMS orally dosed for two consecutive days)

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Stomach Tail Intensity (%) Males and Females	Kidney Tail Intensity (%) Males and Females
Mean	89.53	41.17	55.16	84.92
SD	6.89	14.03	14.23	13.82
n	80	44	59	30
Lower control limit (95% control limits)	79.70	20.78	34.74	72.81
Upper control limit (95% control limits)	99.36	61.56	78.58	97.03

SD = Standard deviation

n = Number of observations

Kidney: Historical control data from experiments performed in Feb 2012 – July 2019

Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019

Conclusions:

When tested in the comet assay, tetraammineplatinum dichloride did not induce an increase in DNA damage in the liver, glandular stomach or duodenum of rats administered up to 1000 mg/kg bw/day by gavage on three consecutive days. A statistically significant and dose-related (p < 0.001 for the trend) increase in DNA damage was seen in kidney cells, but the mean % tail intensity fell within the 95% limits of the historical control data. As such, this finding was considered to be equivocal evidence of a genotoxic effect.

Executive summary:

The potential for tetraammineplatinum dichloride to cause DNA damage was evaluated in a study following OECD 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 250, 500 or 1000 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received two doses of EMS (200 mg/kg bw/day). Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.

There was no increase in % tail intensity in the liver, glandular stomach or duodenum, indicating that the test item is not genotoxic to these tissues.

There was a statistically significant and dose-related increase (p < 0.001) in DNA damage seen in the analysis of the kidney tissue. The tail intensity in animals dosed with 500 mg/kg bw/day was 14.56%, and in animals receiving 1000 mg/kg bw/day was 12.59%. However, these tail intensity values fell within the 95% confidence limits of the historical control data (upper limit 25.55%). As such, this finding was considered to be equivocal evidence of a genotoxic effect. No toxicity was observed in histopathological examination of the kidney tissues, indicating that this can be excluded as an indirect cause of the reported DNA damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

Tetraammineplatinum dichloride was tested (at up to 1 mg/plate) for mutagenic activity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. Dose-related increases in mutant frequency were observed in strains TA1537, TA98 and TA100, both in the presence and absence of metabolic activation (Bootman and Lodge, 1980a).

 

When tested for mutagenic activity in S. typhimurium strains TA98, TA100, TA1535 and TA1538, in the absence of metabolic activation, tetraammineplatinum dichloride induced a positive response in strain TA98 alone (Suraikina et al., 1979).

 

Tetraammineplatinum dichloride was non-mutagenic in a limited Ames test in two strains (TA98 and TA100) of Salmonella typhimurium [the actual doses tested are unclear] (Uno and Morita, 1993).

 

In a limited Ames test, tetraammineplatinum dichloride was not mutagenic in a single strain of Salmonella typhimurium (TA100) when tested solely in the absence of metabolic activation (LeCointe et al., 1979).

 

In an OECD Test Guideline 476 mouse lymphoma assay, tetraammineplatinum (II) hydrogen carbonate induced statistically significant and dose-related increases in the mutant frequency at the TK +/- locus in L5I78Y cells in the presence and absence of metabolic activation, and was considered to be mutagenic under the conditions of the test. However, it was suggested that the mutagenic response was possibly due, or partly due, to a reaction between the test material and the vehicle (DMSO) (Durward, 1998a). In a repeat of this assay, with water as the vehicle, tetraammineplatinum (II) hydrogen carbonate induced a statistically significant dose-related increase in the mutant frequency in L5178Y mouse lymphoma cells in the presence of metabolic activation (Durward, 1998b).

 

In a published study, tetraammineplatinum dichloride was not mutagenic in a gene mutation assay in Chinese hamster ovary cells when tested up to toxic concentrations in the absence of metabolic activation (Johnson et al., 1980).

 

More recently, in an OECD Test Guideline 490 in vitro mammalian cell gene mutation assay, to GLP, tetraammine platinum dichloride induced mutations at the tk locus of L5178Y mouse lymphoma cells when tested at up to cytotoxic concentrations for 3 hours in the absence and presence of S9 and for 24 hours in the absence of S9 (Lloyd, 2017).

 

In an OECD Test Guideline 473 study, conducted to GLP, tetraammineplatinum diacetate did not induce chromosome aberrations in Chinese hamster ovary cells in vitro, both in the absence and presence of metabolic activation (Ciliutti et al., 2007). As part of the same study, tetraammineplatinum diacetate did not induce chromosome aberrations in Chinese hamster ovary cells in vitro, in the presence of metabolic activation (Ciliutti et al., 2008).

 

No increase in sex-linked recessive lethal mutations was observed in the progeny of Drosophila melanogaster following oral administration of tetraammineplatinum dichloride at concentrations of 64 or 320 µg/kg bw/day (Bootman and Lodge, 1980b).

 

Tetraammineplatinum dichloride showed no evidence of clastogenicity in an in vivo assay for chromosome aberrations in bone marrow cells when administered to Chinese hamsters at up to 1000 mg/kg bw/day for 5 consecutive days (Bootman and Rees, 1981).

 

Further, no evidence of clastogenicity was apparent in an in vivo micronucleus assay in mice after a single dose of up to 5000 mg tetraammineplatinum dichloride/kg bw (Bootman and Whalley, 1980).

 

In a combined in vivo micronucleus test and Comet assay in rats, tetraammineplatinum dichloride administered by gavage at doses of 250, 500 or 1000 mg/kg bw/day for three days did not cause an increased incidence of micronucleated polychromatic erythrocytes. Treatment also gave no evidence of DNA damage in the liver, glandular stomach or duodenum when assessed by the Comet procedure. Analysis of the kidney tissue showed evidence of a statistically-significant and dose-related increase in % tail intensity. However, this increase fell within the historical control ranges and was therefore considered as equivocal evidence of a genotoxic effect (Eurlings, 2020).

 

Tetraammine platinum hydrogen carbonate did not induce any marked or toxicologically significant increases in the incidence of cells undergoing unscheduled DNA synthesis in isolated rat hepatocytes following in vivo exposure to 700 or 2000 mg/kg bw for 2 and 16 hours and was considered to be non-genotoxic under the conditions of this study (Durward, 1999).

 

Tetraammineplatinum diacetate and hydrogen carbonate are considered to fall within the scope of the read-across category "tetraammineplatinum(II) salts". See section 13 in IUCLID for full read-across justification report.

 

Several Expert Groups have assessed the toxicity profile of platinum, and various platinum compounds, including the assessment of CMR properties. All reviews have indicated that platinum compounds have been reported to be mutagenic in vitro (DECOS, 2008; EMA, 2008; SCOEL, 2011; WHO, 1991). Cisplatin and related compounds are known DNA-reactive carcinogens and, as these compounds are better investigated due to their pharmaceutical properties, this has been confirmed in vivo. As cisplatin-type substances differ in chemical reactivity (lability of ligands, number of active sites etc.) it is reasonable to expect that not all forms of platinum are carcinogenic (DECOS, 2008). Limited experimental data on reproductive toxicity and carcinogenicity for other platinum compounds give no evidence of activity that would meet classification criteria (DECOS, 2008; SCOEL, 2011).

 

Following the generally positive in vitro results identified for the tetraammineplatinum compounds in various bacterial/mammalian cell mutagenicity assays (supported by some mammalian cell cytogenicity tests) and the unclear in vivo relevance of these in vitro findings, a combined in vivo micronucleus test and Comet assay in rats (with tetraammineplatinum dichloride) did not cause an increased incidence of micronucleated polychromatic erythrocytes and gave no evidence of DNA damage in the liver, glandular stomach or duodenum when assessed by the Comet procedure. However, analysis of the kidney tissue showed evidence of a statistically-significant and dose-related increase in % tail intensity. Nevertheless, this increase fell within the historical control ranges and was therefore considered as equivocal evidence of a genotoxic effect (Eurlings, 2020).

 

In order to assess the potential to induce gene mutations in vivo, a transgenic rodent (TGR) gene mutation assay (according to OECD Test Guideline 488) is proposed.

References

DECOS (2008). Dutch Expert Committee on Occupational Standards. Platinum and Platinum Compounds. Health-based recommended occupational exposure limit. Gezondheidsraad, 2008/12OSH. https://www.gezondheidsraad.nl/en/publications/gezonde-arbeidsomstandigheden/platinum-and-platinum-compounds-health-based-recommended

 

EMA (2008). European Medicines Agency. Guideline on the specification limits for residues of metal catalysts or metal reagents. Committee for Medicinal Products for Human Use (CHMP). EMEA/CHMP/SWP/4446/2000. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003586.pdf

 

SCOEL (2011). Recommendation from the Scientific Committee on Occupational Exposure Limits for platinum and platinum compounds. SCOEL/SUM/150. http://ec.europa.eu/social/BlobServlet?docId=7303&langId=en

 

WHO (1991). World Health Organization. Platinum. International Programme on Chemical Safety. Environmental Health Criteria 125. http://www.inchem.org/documents/ehc/ehc/ehc125.htm#SectionNumber:7.4

Justification for classification or non-classification

Based on the existing data set, tetraammineplatinum dichloride does not currently meet the criteria for classification as a germ cell mutagen (category 1A/1B or 2).