Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a well-conducted OECD Test Guideline 471 study, tetraammineplatinum(II) 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 (Verspeek-Rip, 2004).

 

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).

 

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(II) 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(II) diacetate did not induce chromosome aberrations in Chinese hamster ovary cells in vitro, in the presence of metabolic activation (Ciliutti et al., 2008).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 to 9 September 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study, to GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
substance was mixed with 3 ml surface agar, whereas the guideline recommends 2 ml
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
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:
In a GLP study performed according to OECD Test Guideline 471, tetraammineplatinum diacetate was mutagenic in Salmonella typhimurium strains TA98 and TA1537 and Escherichia coli strain WP2uvrA when tested aat up to 5 mg/plate in the presence and absence of metabolic activation.
Executive summary:

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

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data were identified for tetraammineplatinum(II) diacetate. However, a number of in vivo studies on related tetraammineplatinum(II) salts were available.

 

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

 

Tetraammineplatinum(II) 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 with tetraammineplatinum(II) dichloride in an in vivo micronucleus assay in mice after a single dose of up to 5000 mg/kg bw (Bootman and Whalley, 1980).

 

Tetraammineplatinum(II) 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).

 

Further in vivo genotoxicity testing is proposed.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: Read-across from study conducted on a member of the "tetraammineplatinum(II) salts" category, tetraammineplatinum dichloride
Adequacy of study:
weight of evidence
Study period:
17 March 1980 to 9 May 1980
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Method used was similar to guideline, though this type of study is no longer recommended for regulatory testing. Nevertheless, the data from this study is useful in the overall weight of evidence.
Remarks:
Study conducted on read-across compound
Justification for type of information:
Tetraammineplatinum dichloride is 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.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Deviations:
no
Principles of method if other than guideline:
Spencer and Stern (1948) Genetics 33, 43-74
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Males: Oregon; K Females: Muller-5
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: in-house; derived from cultures obtained from the Genetics Department, University of Edinburgh
- Age at study initiation:  1 week
- Assigned to test groups randomly: [no/yes, under following basis: ] yes
- Fasting period before study: 2-3 hr
- Housing: glass jars
- Diet (e.g. ad libitum): Drosophila medium with powdered yeast sprinkled on top


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25

IN-LIFE DATES: From: To:

Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: 1% sucrose
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 64 or 320 µg/ml
- Type and concentration of dispersant aid (if powder): placed on filter paper
:

Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Daily on each day of treatment
Flies remove to clean jars without normal feeding solution for 2-3 hours then given the treated filter papers for 17 hr before being removed to jars containing normal nutrient medium

Duration of treatment / exposure:
17 hr/day for 3 days
Frequency of treatment:
17 hr/day for 3 days
Post exposure period:
24 hr before beginning mating
Remarks:
Doses / Concentrations:
64 or 320 µg
Basis:
nominal in diet
No. of animals per sex per dose:
50 males selected for mating
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): recommended substance
- Route of administration: oral
- Doses / concentrations: 50 μg/ml in 1% sucrose solution

Tissues and cell types examined:
Presence or absence of red-eyed males
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: as the result of a range-finding study to find a top dose in which survival was at least 50% without markedly impaired fertility

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Treated males mated with s sets of virgin females for 3 days each, before being placed with new females for a further 6 days. The offspring (F1 generation) were then mated together to produce the F2 generation. Cultures considered to carry X-lethals or suspect lethals/semi-lethals were retested using red-eyed females from the F2 generation.

METHOD OF ANALYSIS: examination by eye for the absence or presence of red-eyed flies in F2 and F3 generations.

Evaluation criteria:
Vials containing 3 or more red-eyed (wild-type) males were scored as non-lethal. F2 cultures having at least 11 phenotypically M-5 males and no wild-type males were considered to show the X-lethal mutation. Cultures having less than 11 M-5 males or containing 1-2 wild-type males were regarded as suspect semi-lethals or lethals
Statistics:
Fisher’s exact probability test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY 1
- Dose range: 0.32, 1.6, 8.0, 40 and 200 mg/ml
- Solubility: suspension
- Clinical signs of toxicity in test animals: 83-90% on the three top doses died, 40-60% died on the two lower doses
- Evidence of cytotoxicity in tissue analyzed: not applicable
- Rationale for exposure: to determine dose level giving at least 50% survival


RESULTS OF DEFINITIVE STUDY
The test substance did not induce sex-linked recessive lethal mutations (see below)
- Appropriateness of dose levels and route: appropriate
- Statistical evaluation: see below


Conclusions:
Tetraammineplatinum(II) chloride did not induce sex-linked recessive lethal mutations in the progeny of Drosophila melanogaster following oral exposure to up to 320 µg/kg bw/day for 3 consecutive days.
Executive summary:

Tetraammineplatinum(II) chloride was assessed for mutagenic potential in an assay for sex-linked recessive lethal mutations in Drosophila melanogaster.

Males were given access to a 1% sucrose “feed” for 17 hr daily on three consecutive days at tetraammineplatinum(II) chloride doses of about 64 or 320 µg/kg bw/day. Twenty four hr after treatment they were mated with groups of untreated virgin females so that each stage of the spermatogenic cycle was sampled. Cultures having at least 11 phenotypically M-5 males and no wild-type males were considered to show the X-lethal mutation and were collected and mated within the treatment group and the resulting progeny were scored for the presence or absence of red-eyed (wild-type) males. All cultures having X-lethal mutations or suspected lethal/semi-lethal mutations were subjected to a further round of mating to confirm the mutations.

The test compound did not increase the mutation frequency when compared to vehicle treated controls. In this assay, tetraammineplatinum(II) chloride showed no evidence of sex-linked recessive lethal mutations in D. melanogaster.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

Only certain in vitro and no in vivo genotoxicity data were identified for tetraammineplatinum(II) diacetate. However, a number of genotoxicity studies (both in vitro and in vivo) were identified for tetraammineplatinum(II) dichloride and hydrogen carbonate. Tetraammineplatinum dichloride 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.

 

The genotoxic potential of tetraammineplatinum(II) 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 S. typhimurium strain TA100 and E. coli strain WP2uvrA. Triplicate cell cultures were exposed to tetraammineplatinum(II) 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 S9 (alongside appropriate vehicle and positive controls), and were incubated for 48 hours at 37 °C before being inspected for signs of cytotoxicity and for the presence of revertant colonies. Significant cytotoxicity was not observed for any of the tested strains, at any of the tested concentrations. Significant, dose-related increases in the number of observed revertant colonies were seen for S. typhimurium strains TA98 (up to 2.3 -fold in the presence of S9) and TA1537 (up to 13 -fold and 18 -fold in the absence and presence of S9 respectively), and E. coli strain WP2uvrA (up to 3.2 -fold and 6 -fold in the absence and presence of S9 respectively). No significant, dose-related increases were seen in S. typhimurium strain TA98 in the absence of S9, or in TA100 and TA1535 (both in the absence and presence of S9). Under the conditions of this assay, tetraammineplatinum(II) diacetate showed mutagenic potential (Verspeek-Rip, 2004).

 

Tetraammineplatinum(II) hydrogen carbonate (in DMSO) was tested in an in vitro mammalian cell gene mutation assay in accordance with OECD Test Guideline 476. L5178Y mouse lymphoma cells were treated - both in the presence and absence of S9 - with 222, 444, 888, 1775 or 3550 µg/mL in one experiment and, because of inconsistent test material toxicity, with 25, 50, 100, 200 and 300 µg/mL in another. Statistically significant dose-related increases in mutant frequency in the absence and presence of S9 were seen in both experiments. Therefore tetraammineplatinum(II) hydrogen carbonate was considered to be mutagenic to L5178Y cells under the conditions of the test (Durward, 1998a). However, it was suggested that the mutagenic response was possibly at least partly due to a reaction between the test material and the vehicle (DMSO), and so another study of this type, using water as a solvent, was performed.

 

In an analogous study, L5178Y mouse lymphoma cells were treated with tetraammineplatinum(II) hydrogen carbonate (in water) at concentrations of 62.5, 125, 250, 500, 750 or 1000 µg/mL both in the presence and absence of S9; a statistically significant dose-related increase in the mutant frequency was seen in the presence of S9. Therefore tetraammineplatinum(II) hydrogen carbonate was considered to be mutagenic to L5178Y cells under the conditions of the test (Durward, 1998b).

 

In a study conducted in a similar manner to OECD Test Guideline 476, the ability of tetraammineplatinum dichloride to induce gene mutations in the Chinese hamster ovary cell line was assessed (in the absence of a metabolic activation system). The maximum number of mutants reported was 17 x 1,000,000 cells (compared to a range of less than 1 to 15 in controls per 1,000,000 cells, and an average in these cells of 3.3 mutants x 1,000,000 cells) and the slope of the concentration-dependent mutation-induction curve was (2.52±2.3) x 0.001 mutants/1,000,000 cells/μM (with a 50% probability of it being significantly greater than zero). In conclusion, the test substance did not induce gene mutations in the Chinese hamster ovary cell line when tested up to toxic concentrations in the absence of metabolic activation (Johnson et al., 1980).

 

In a recent 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 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 (Lloyd, 2017).

The clastogenicity of tetraammineplatinum(II) diacetate was assessed in an in vitro study on Chinese hamster ovary (CHO) 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 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 S9. 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(II) diacetate was not clastogenic in CHO cells in vitro (Ciliutti et al., 2007).

 

The clastogenic potential of tetraammineplatinum(II) diacetate was confirmed in a subsequently conducted in vitro study on CHO 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 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(II) diacetate was not clastogenic in CHO cells in vitro, in the presence of S9 (Ciliutti et al., 2008).

 

Tetraammineplatinum(II) chloride was assessed for mutagenic potential in an assay for sex-linked recessive lethal mutations in Drosophila melanogaster. Males were given access to a 1% sucrose “feed” for 17 hr daily on three consecutive days at tetraammineplatinum(II) chloride doses of about 64 or 320 µg/kg bw/day. Twenty four hr after treatment they were mated with groups of untreated virgin females so that each stage of the spermatogenic cycle was sampled. Cultures having at least 11 phenotypically M-5 males and no wild-type males were considered to show the X-lethal mutation and were collected and mated within the treatment group and the resulting progeny were scored for the presence or absence of red-eyed (wild-type) males. All cultures having X-lethal mutations or suspected lethal/semi-lethal mutations were subjected to a further round of mating to confirm the mutations. The test compound did not increase the mutation frequency when compared to vehicle treated controls. In this assay, tetraammineplatinum(II) chloride showed no evidence of sex-linked recessive lethal mutations in D. melanogaster (Bootman and Lodge, 1980).

 

Tetraammineplatinum(II) chloride was studied for the ability to induce micronuclei in bone marrow cells in mice, in a study conducted largely in accordance to OECD Test Guideline 474. Animals (5/sex/group) received tetraammineplatinum(II) chloride by oral gavage at a single dose of 200, 500 or 5000 mg/kg bw and bone marrow cells from the femurs were sampled 24 hr after treatment. Two thousand cells per animal were scored for micronuclei and the ratio of polychromatic to mature cells was calculated as a measure of toxicity. The test compound did not increase the frequency of micronucleated cells when compared to the vehicle control. In contrast, there was a statistically significant increase in micronucleus frequency induced by the positive control. In is concluded that tetraammineplatinum(II) chloride did not increase the incidence of micronuclei in mice 24-hr after a single dose of up to 5000 mg/kg bw (Bootman and Whalley, 1980).

 

In a well-conducted study, equivalent to OECD Test Guideline 475, tetraammineplatinum(II) chloride hydrate was assessed for its ability to induce chromosome aberrations in the bone marrow cells of Chinese hamsters. Groups of six males received tetraammineplatinum(II) chloride hydrate by oral gavage at doses of 40-1000 mg/kg bw/day for 5 days, and the bone marrow cells harvested from the femurs 24 hr after the last treatment. After fixing and staining, 100 metaphases per dose were scored for numbers and all types of aberrations (gaps were excluded from the analysis of total aberrations). No increase in aberrations was detected in animals treated with the test material compared to the frequency observed in animals treated with vehicle alone. In contrast, there was a statistically significant increase in aberration frequency after treatment with the positive control. It is concluded that tetraammineplatinum(II) chloride hydrate did not induce chromosome aberrations in Chinese hamsters treated at up to 1000 mg/kg bw daily for 5 days (Bootman and Rees, 1981).

 

The potential of tetraammineplatinum(II) hydrogen carbonate to induce DNA repair (unscheduled DNA synthesis) in isolated rat hepatocytes following oral administration to rats was investigated. The method used was designed to comply with OECD Test Guideline 486, and followed the recommendations of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993). Animals were given gavage doses of 2000 mg/kg bw or 700 mg/kg bw and liver cells were examined after approximately 2 or 16 hours (stained slides were scored using a microscope and an automated image analysis system). The method used for scoring the hepatocytes was an area counting method which complies with the UKEMS guidelines. There was no toxicologically significant increase in the incidence of unscheduled DNA synthesis in animals dosed with test material at either time point. As such tetraammineplatinum hydrogen carbonate was considered to be non-genotoxic under the conditions of the test (Durward, 1999).

 

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).

 

Despite the generally positive in vitro results identified for the platinum compounds in various bacterial/mammalian cell mutagenicity assays (supported by some mammalian cell cytogenicity tests), the in vivo relevance of these in vitro findings remains unclear. Indeed, the available in vivo data returned mostly negative results, including studies on closely-related tetraammineplatinum(II) salts. However, some of the identified studies might not be considered sufficiently robust (according to ECHA standards) to override the in vitro mutagenicity findings (e.g. a sex-linked recessive lethal test in Drosophila melanogaster (OECD TG 477, performed with tetraammineplatinum(II) dichloride) and a liver unscheduled DNA synthesis assay (OECD TG 486, performed with tetraammineplatinum(II) hydrogen carbonate)). Additional in vivo testing of certain platinum compounds has been proposed to further elucidate the in vivo relevance of the in vitro findings.

 

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(II) diacetate does not currently meet the criteria for classification as a germ cell mutagen under EU CLP criteria (EC 1272/2008). However, this conclusion should be revisited when the results of the planned in vivo studies are available.