Hexachloroplatinic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a good-quality OECD Test Guideline 471 study, to GLP, hexachloroplatinum(IV) solution induced reverse mutations in Salmonella typhimurium strains TA98 and TA100 and in Escherichia coli WP2 uvrA under the experimental conditions (Scarcella, 2004).

 

In another well-conducted OECD Test Guideline 471 study, to GLP, dihydrogen hexachloroplatinate(IV) solution was equivocally mutagenic in S. typhimurium strain TA100 and not mutagenic in strains TA1535, TA1537 and TA98 or in E. coli, when tested in the presence and absence of metabolic activation (Verspeek-Rip, 2002).

 

In an OECD Test Guideline 476 study, to GLP, dihydrogen hexachloroplatinate(IV) solution was mutagenic at the TK locus of mouse lymphoma (L5178Y) cells when tested in the absence and presence of S9 (Verspeek-Rip, 2003).

 

No mammalian cell cytogenicity data are available for hexachloroplatinic acid. Further in vitro testing in considered unnecessary in the light of the proposed in vivo genotoxicity testing.

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

Study period:

4 September - 26 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conduced to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (S. typhimurium) and tryptophan (E.coli) loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 from male Sprague-Dawley rats given prior treatment with phenobarbital abd betanaphthoflavone

Test concentrations with justification for top dose:

Two main experiments were performed:

In experiment 1 (plate incorporation method) the test item was assayed in TA1535 at 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 ug/plate, and in TA1537, TA98, TA100 and WP2uvra at 1.25, 2.5, 5.0, 10.0, 20.0 and 40.0 ug/plate (without S9). In all five strains it was tested at 10.0, 20.0, 40.0, 80.0, 160.0 ug/plate (with S9); TA98 and TA100 were also tested at 320 ug/plate (with S9).

In experiment 2 (pre-incubation method) the test item was assayed in TA1535, TA98 and TA100 at 0.313, 0.625, 1.25, 2.5, 5.0 and 10.0 ug/plate (in the absence of S9); TA100 was also tested at 20.0 and 40.0 ug/plate. TA1537 was tested at 0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 ug/plate (without S9).In the presence of S9, all four Salmonella strains were tested at 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 ug/plate; TA1537 and TA100 were also tested at 1.25 and 160 ug/plate, and TA98 additionally at 0.625 ug/plate (with S9). WP2uvra was not used in experiment 2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: soluble at 50.0 mg/ml. Since 100 ul of test item soluition used in the preparation of each plate, this permitted a max. conc. of 5000 ug/plate in toxicity test.

Untreated negative controls:

yes

Remarks:

Dimethylsulphoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium strains TA1535, TA1537, TA100, TA98 and Escherichia coli WP2uvrA were used in experiment 1 (plate incorporation method) and all four S. typhimurium strains in experiment 2 (pre-incubation method), excluding E. Coli WP2uvrA. 3 replicate plates. Dose range finding study, and two main experiments (1 and 2) only.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose-levels or at the highest practicable dose-level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels.

Number of cells evaluated per dose group not given in report.

Statistics:

Not reported

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

dose-related increases, at least two fold

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

dose-related increase in revertant numbers (experiments 1 & 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

dose-related increase in revertant numbers (experiments 1 & 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

experiments 1 & 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

experiments 1 & 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

experiments 1 & 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

experiments 1 & 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
positive

In an OECD Test Guideline 471 study, to GLP, hexachloroplatinum(IV) solution induced reverse mutations in Salmonella typhimurium strains TA98 and TA100 and in Escherichia coli WP2 uvrA under the experimental conditions.

Executive summary:

In an OECD Test Guideline 471 study, conducted according to GLP, hexachloroplatinum(IV) solution was assessed for its ability to induce gene mutations in strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli (WP2 uvrA). The test was performed in two independent experiments, with dose levels determined following an initial toxicity test, both in the absence and presence of metabolic activation (S9) using liver fraction from rats pre-treated with phenobarbitone and beta-naphthoflavone.

 

In experiment 1, dose-related increases in revertant numbers, which were at least two-fold the control values, were observed in WP2 uvrA both in the absence and presence of S9. Dose-related increases in revertant numbers were also observed in TA98 and TA100 tester strains in the presence of S9.

 

In experiment 2, conducted using the four S. typhimurium strains, large dose-related increases in revertant numbers were observed at higher dose levels in TA98 in the presence of S9. Dose-related and reproducible increases in revertant numbers were also observed in TA100 tester strain in the presence of S9. Although these increases did not reach two-fold the control values they were considered clear evidence of a mutagenic effect.

 

It was concluded that hexachloroplatinum(IV) solution was mutagenic in S. typhimurium strains TA98 and TA100 and in E. coli WP2 uvrA under the experimental conditions.

Reference 2

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:

17 October 2002 - 04 November 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (S.typhimurium); Tryptophan (E.coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

In the dose range finding study with S. typhimurium TA100 and E.coli WP2uvrA only, levels of 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate were used. In experiment 1 the five strains were tested at 0.1, 0.3, 1, 3, 10 and 33 ug/plate (without S9) and at 1, 3, 10, 16, 33 and 100 ug/plate (with S9). In experiment 2, the five strains were tested at 0.3, 1, 3, 10, 16 (TA100 only), and 33 ug/plate (in the absence of S9) and 3, 10, 16, 33, 100 ug/plate (in the presence of S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not stated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

TA100 - without S9

Positive control substance:

methylmethanesulfonate

Positive controls:

yes

Remarks:

TA98 - without S9

Positive control substance:

other: daunomycin

Positive controls:

yes

Remarks:

TA1535 - without S9

Positive control substance:

sodium azide

Positive controls:

yes

Remarks:

TA1537 - withot S9

Positive control substance:

9-aminoacridine

Positive controls:

yes

Remarks:

WP2uvrA - without S9

Positive control substance:

4-nitroquinoline-N-oxide

Positive controls:

yes

Remarks:

all strains - with S9

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

In a dose range finding study S. typhimurium TA100 and E.coli WP2uvrA only were teasted at levels of 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate were used (with and without S9). In experiment 1 the five strains were tested at 0.1, 0.3, 1, 3, 10, and 33 ug/plate (without S9) and at 1, 3, 10, 16, 33 and 100 ug/plate (with S9). In experiment 2, the five strains were tested at up to 33 ug/plate in the absence of S9 and up to 100 ug/plate in the presence of S9.

3 replicate plates

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

108 cells/plate (0.1 ml of fresh bacterial culture containing 109 cells/ml) were assessed

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

experiments 1 & 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

dose range finding study

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: all other strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In strain TA100, 1.8 and 1.7-fold increases in the number of revertant colonies compared to the solvent control were observed in the absence of S9-mix in experiments 1 and 2, respectively. These increases were not-dose-related, were observed at the highest cytotoxic dose level only (33 ug/plate) and not observed in the dose range finding study. However, since the increases were observed in two independently repeated experiments and outside the reported historical control data range, these increases were considered by the investigators to be equivocal.

In the dose range finding study in strain TA100, a 2.3-fold increase in the number of revertant colonies compared to the solvent control was observed in the presence of 5% S9. No verification of this result was observed either in experiment 1 in the presence of 5% S9 or after modification in the concentration of S9 to 10% in experiment 2. Since the mutagenic effect observed in the dose range finding study was more than two-fold the solvent control and outside the reported historical control data range, this increase was considered by the investigators to be equivocal.

All other bacterial strains showed a negative response over the entire dose range, both with and without S9, i.e. no dose-related, two-fold increase in the number of revertants in the two independently repeated experiments.

Conclusions:

Interpretation of results (migrated information):
ambiguous

In an OECD Test Guideline 471 study, to GLP, dihydrogen hexachloroplatinate(IV) solution was equivocally mutagenic in Salmonella typhimurium strain TA100 and not mutagenic in strains TA1535, TA1537 and TA98 or in Escherichia coli, when tested in the presence and absence of metabolic activation.

Executive summary:

The mutagenic potential of dihydrogen hexachloroplatinate (IV) solution was assessed in a reverse mutation assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assessed in four Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and in Escherichia coli WP2 uvrA, in an attempt to detect both base-pair substitution and frameshift mutations. The test was performed in two independent experiments in the presence and absence of metabolic activation from Arochlor-1254 induced rat liver (S9).

 

In a dose range finding test, dihydrogen hexachloroplatinate (IV) solution was tested up to 5000 ug/plate in the absence and presence of 5% S9 mix with strains TA100 and WP2urvA. Based on the results of this test, in experiment 1 the test substance was tested up to 33 and 100 ug/plate in the absence and presence of 5% S9 mix, respectively. In experiment 2 the test substance was tested up to 33 and 100 ug/plate in the absence and presence of 10% S9 mix, respectively. Toxicity was observed in all tested strains.

 

In strain TA100, 1.8- and 1.7-fold increases in the number of revertant colonies were observed in the absence of S9 in the first and second experiments, respectively. These increases were not dose-related, were observed at the highest cytotoxic dose level only, and were not apparent in the dose-range finding study. However, since the increases were observed in two independently repeated experiments and outside the range of historical control data, these increases were considered by the investigators to be equivocal.

 

A 2.3-fold increase in the number of revertant colonies was observed in the presence of S9 in the dose range finding study in strain TA100, though this result was not verified in experiments 1 and 2. However, since this mutagenic effect was more than two-fold and outside historical control data range, it was also considered to be equivocal.

 

All other bacterial strains showed a negative response over the entire dose range in both experiments.

 

It was concluded that dihydrogen hexachloroplatinate (IV) solution was equivocally mutagenic in the S. typhimurium strain TA100, and not mutagenic in E. coli.

Reference 3

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:

12 February 2003 - 04 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Based on OECD test guideline 476

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine-kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

In the dose range finding test, cells were treated with a test substance concentration range of 10 to 5000 ug/ml.
In the mutation test, cells were tested at 1, 3, 10, 18, 24, 32, 56 and 75 µg/ml exposure medium (without S9) and 10, 18, 32, 42, 56, 75, 100 and 133 µg/ml exposure medium (with S9).

Vehicle / solvent:

ethanol

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Remarks:

without S9

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 2 mM

Positive controls:

yes

Remarks:

with S9

Positive control substance:

N-dimethylnitrosamine

Remarks:

Migrated to IUCLID6: 0.5 mM

Evaluation criteria:

A test substance was considered positive (mutagenic) in the mutation assay if:
a) It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; or
Negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b) The results were confirmed in an independently repeated test.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

With metabolic activation: The test substance induced a 12-fold, dose-related, increase in the mutant frequency at the TK locus, and 16- and 11-fold increases in the mutant frequency of the small and large colonies, respectively, compared to the solvent controls. The actual cell survival at the highest test substance concentration was recuced by 83% compared to the solvent controls.

Without metabolic activation: The test substance induced an up to 28-fold, dose-related, increase in the mutant frequency at the TK locus, and a 24-fold increase in the mutant frequency of the large colonies compared to the solvent controls. A dose-related increase in the mutant frequency of the small colonies by the test substance was observed, however, since the amount of small colonies in the solvent controls was zero, no statisitcal results could be calculated. The actual cell survival at the highest test substance concentration was recuced by 95% compared to the solvent controls.

Conclusions:

Interpretation of results (migrated information):
positive

In an OECD Test Guideline 476 study, to GLP, dihydrogen hexachloroplatinate(IV) solution was mutagenic at the TK locus of mouse lymphoma (L5178Y) cells when tested in the absence and presence of S9.

Executive summary:

The ability of dihydrogen hexachloroplatinate(IV) solution to induce forward mutations at the thymidine-kinase (TK) locus in mouse lymphoma (L5178Y) cells was evaluated in an OECD Test Guideline 476 study, conducted according to GLP, both in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9 -mix). A range-finding study was conducted to set dose levels for the subsequent mutation assay.

 

In the main experiment, dihydrogen hexachloroplatinate(IV) solution was tested up to concentrations of 75 and 133 μg/mL, with an incubation time of 3 hours, in the absence and presence of S9, respectively.

 

The test material induced clear, dose-related, increases in the mutant frequency at the TK locus in the absence and presence of S9 (respective increases of 28.5- and 11.9-fold at the highest concentrations). The cell survival (compared to the solvent control) was reduced by 95% and 83% at the highest tested concentrations in the absence and presence of S9 -mix, respectively.

 

Since a clear, dose dependent, positive response was observed in the mutant frequency at the TK locus after treatment, no repeat assay was performed.

 

It was concluded that dihydrogen hexachloroplatinate(IV) solution was mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genotoxicity of diammonium hexachloroplatinate, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes and to cause DNA damage, was assessed in a combined study following OECD 474 and 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The highest-tested dose was limited by clinical signs of toxicity, including mortalities, observed in a dose range finding study. Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues and micronuclei were analysed in bone marrow cells. 

There was no increase in the number of micronucleated polychromatic erythrocytes in any treatment group. There was no increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item was not genotoxic to these tissues. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate -and via read-across also hexachloroplatinic acid- was concluded to be non-genotoxic in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May 2020 - 09 Jul 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016.

Deviations:

no

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: AI2707.
- Expiration date of the lot/batch: 25 August 2020 (from CoA).
- Purity: 99%.
- Purity test date: CoA issued 22 January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.

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: 150 ± 7.8 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: Approx. 20 Mar 2020 (6 weeks before experimental start date).
To: 10 Jun 2020.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: 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 109%, 103% and 108% of the nominal values for group 2, group 3 and group 4 (i.e. 37.5, 75 and 105 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation ≤ 10%) of the test item in vehicle was confirmed.
- Amount of vehicle (if gavage or dermal): dosing volume was 10 mL/kg bw
- Stability of test item in vehicle: stability of test item suspended in vehicle demonstrated for 4 hours at room temperature under normal laboratory conditions(sufficient for the dosing of all test animals), after which unused test item formulations were discarded.
-A separate study was performed to develop and to validate an analytical method for the
quantitative analysis of Diammonium hexachloroplatinate (CAS 16919-58-7) in vehicle (corn
oil) (Charles River Labs, Test Facility Study No. 20213554). The analytical method was validated for the following parameters:
• Specificity
• Calibration curve
• Accuracy and Repeatability
• Limit of quantification
• Stability of the analytical system and end solutions
• Stability of stock solutions
For Concentration and Homogeneity Analysis, storage conditions were at room temperature and the Acceptance Criteria for concentration (mean sample concentration results within or equal to
± 15% suspensions of theoretical concentration) and for homogeneity (relative standard deviation (RSD) of concentrations of +/- 10% for each group) were met. For Stability Analysis (4 h at room temperature), the Acceptance Criteria (Mean sample concentration results after storage that is within or equal to ±10% of the initial mean sample concentration results) were met too. The validated ICP-MS/MS method is thus considered suitable for the quantitative analysis of the
test item, based on platinum, in vehicle, in the test item target concentration range of
0.1 – 200 mg test item/g.

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.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Test item-related mortality was observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 200 mg/kg bw, and one animal of each sex received 300 mg/kg bw/day. Clinical signs of toxicity (ataxia, lethargy, hunched posture, rough coat and diarrhoea) were observed at 150 mg/kg bw/day, which was determined to be the maximum tolerated dose.

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 pre-coated comet slide (Trevigen), which was then incubated for 10 - 21 minutes in the refrigerator. Three slides per tissue were prepared.

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:

150 comets were examined per sample using an IV image analysis system. Only horizontal comets, oriented with the head on the left and the tail on the right, were scored. Cells that showed overlap or were not sharp were not scored.


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, and direct or indirect evidence supportive of exposure of, or toxicity to, the target tissues was demonstrated, the test item was considered negative. If the data precluded making a conclusion of clearly positive or negative, the result was concluded as equivocal.

Statistics:

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

A test item is considered positive in the comet assay 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 percentage Tail Intensity is detected 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 comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in percentage Tail Intensity is detected 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.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Kidney: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Liver: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Duodenum: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

One high-dose animal died after the first dose, and was replaced by an additional animal. Clinical signs of toxicity were observed in the high-dose group: hunched posture (4/5 animals), lethargy (5/5 animals) and diarrhoea (1/5 animals).

Platinum was quantifiable in plasma samples from high-dose (150 mg/kg bw/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 hours after the third dose. Therefore it was confirmed that there was systemic exposure to the test item. No test item was detected in the animals dosed with vehicle.

Group mean % tail DNA for the different tissues analyses (mean and standard deviation)
liver	tail intensity (%)	SD
vehicle control	4.54	0.63
test item 37.5 mg/kg	4.70	0.74
test item 75 mg/kg	4.28	0.79
test item 150 mg/kg	3.76	0.23
EMS 200 mg/kg	82.11*	6.52
* significantly different (p<0.001) compared to corresponding vehicle control group
duodenum	tail intensity (%)	SD
vehicle control	7.57	1.48
test item 37.5 mg/kg	6.64	1.30
test item 75 mg/kg	5.8	0.93
test item 150 mg/kg	6.15	0.78
EMS 200 mg/kg	46.84*	4.76
* significantly different (p<0.001) compared to corresponding vehicle control group
stomach	tail intensity (%)	SD
vehicle control	6.32	1.57
test item 37.5 mg/kg	6.47	0.33
test item 75 mg/kg	4.22	0.83
test item 150 mg/kg	5.44	1.31
EMS 200 mg/kg	53.24*	4.73
* significantly different (p<0.001) compared to corresponding vehicle control group
kidney	tail intensity (%)	SD
vehicle control	4.62	0.84
test item 37.5 mg/kg	4.96	1.84
test item 75 mg/kg	5.11	1.00
test item 150 mg/kg	5.16	0.59
EMS 200 mg/kg	86.05*	4.45
* significantly different (p<0.001) compared to corresponding vehicle control group

Historical data Comet assay Negative control
	Liver	Duodenum	Stomach	Kidney
	Tail Intensity (%)	Tail Intensity (%)	Tail Intensity (%)	Tail Intensity (%)
	Males and Females	Males and Females	Males and Females	Males and Females
Mean	2.4	4.3	3.5	9.1
SD	1.6	2.0	1.8	7.9
n	34	19	22	9
Lower control limit (95% control limits)	-0.8	0.3	0.0	-6.3
Upper control limit (95% control limits)	5.6	8.2	7.0	24.5
SD = Standard deviation
n = Number of observations
Kidney: Historical control data from experiments performed in Feb 2012 – June 2020
Liver, Stomach, Duodenum: Historical control data from experiments performed in July 2017 – June 2020
Historical data Comet assay Positive control (200 mg/kg bw EMS orally dosed for two consecutive days)
	Liver	Duodenum	Stomach	Kidney
	Tail Intensity (%)	Tail Intensity (%)	Tail Intensity (%)	Tail Intensity (%)
	Males and Females	Males and Females	Males and Females	Males and Females
Mean	87.7	45.4	55.3	83.3
SD	6.7	12.1	11.6	11.8
n	33	19	22	9
Lower control limit (95% control limits)	74.5	21.7	32.6	60.2
Upper control limit (95% control limits)	100.9	69.1	78	106.4
SD = Standard deviation
n = Number of observations
Kidney: Historical control data from experiments performed in Feb 2012 – June 2020
Liver, Stomach, Duodenum: Historical control data from experiments performed in July 2017 – June 2020

Conclusions:

When tested in the comet assay, diammonium hexachloroplatinate did not induce DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 150 mg/kg bw/day by gavage on three consecutive days. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.

Executive summary:

The potential for diammonium hexachloroplatinate 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 37.5, 75 or 150 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 statistically significant increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item was not genotoxic to these tissues. 

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May 2020 - 09 Jul 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: AI2707.
- Expiration date of the lot/batch: 25 August 2020 (from CoA).
- Purity: 99%.
- Purity test date: CoA issued 22 January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.

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: 150 ± 7.8 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: Approx. 20 Mar 2020 (6 weeks before experimental start date).
To: 10 Jun 2020.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: 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 109%, 103% and 108% of the nominal values for group 2, group 3 and group 4 (i.e. 37.5, 75 and 105 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation ≤ 10%) of the test item in vehicle was confirmed.
- Amount of vehicle (if gavage or dermal): dosing volume was 10 mL/kg bw
- Stability of test item in vehicle: stability of test item suspended in vehicle demonstrated for 4 hours at room temperature under normal laboratory conditions(sufficient for the dosing of all test animals), after which unused test item formulations were discarded.
-A separate study was performed to develop and to validate an analytical method for the
quantitative analysis of Diammonium hexachloroplatinate (CAS 16919-58-7) in vehicle (corn
oil) (Charles River Labs, Test Facility Study No. 20213554). The analytical method was validated for the following parameters:
• Specificity
• Calibration curve
• Accuracy and Repeatability
• Limit of quantification
• Stability of the analytical system and end solutions
• Stability of stock solutions
For Concentration and Homogeneity Analysis, storage conditions were at room temperature and the Acceptance Criteria for concentration (mean sample concentration results within or equal to ± 15% suspensions of theoretical concentration) and for homogeneity (relative standard deviation (RSD) of concentrations of +/- 10% for each group) were met. For Stability Analysis (4 h at room temperature), the Acceptance Criteria (Mean sample concentration results after storage that is within or equal to ±10% of the initial mean sample concentration results) were met too. The validated ICP-MS/MS method is thus considered suitable for the quantitative analysis of the test item, based on platinum, in vehicle, in the test item target concentration range of 0.1 – 200 mg test item/g.

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.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Test item-related mortality was observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 200 mg/kg bw, and one animal of each sex received 300 mg/kg bw/day. Clinical signs of toxicity (ataxia, lethargy, hunched posture, rough coat and diarrhoea) were observed at 150 mg/kg bw/day, which was determined to be the maximum tolerated dose.

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.

The number of micronucleated polychromatic erythrocytes was initially counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).Slides were scored at a magnification of 1000x.

The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

The test item was considered positive if all of the following criteria were met:
a) at least one treatment group showed a statistically significant increase in frequency of micronucleated polychromatic erythrocytes.
b) the increase was dose related.
c) the results were outside the 95% confidence limits of the historical control data.

If none of the above criteria were met, and bone marrow exposure to the test item occurred, the substance was considered negative.

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

Statistics:

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

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.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One high-dose animal died after the first dose, and was replaced by an additional animal. Clinical signs of toxicity were observed in the high-dose group: hunched posture (4/5 animals), lethargy (5/5 animals) and diarrhoea (1/5 animals).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Platinum was quantifiable in plasma samples from high-dose (150 mg/kg bw/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 hours after the third dose. Therefore it was confirmed that the bone marrow was 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.

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

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	4	4.6	± 1.1	1.11	± 0.16
			2	5
			3	3
			4	5
			5	6
2	test item	37.5	6	0	1.6	± 1.7	1.22	± 0.06
			7	0
			8	2
			9	2
			10	4
3	test item	75	11	0	2.4	± 1.5	1.18	± 0.18
			12	2
			13	3
			14	4
			15	3
4	test item	150	16	2	3.2	± 2.2	1.0	± 0.08
			17	0
			18	4
			19	5
			21	5
6	Cyclophosphamide	19	29	8	10.8	± 1.8 (4)	0.91	± 0.07
			30	12
			31	12
			32	10
			33	12

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 (Students t test, P < 0.001).

 

Dose-response relationship & statistics:
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 Student t-test

 

Distribution historical control data from experiments performed between June 2017 and June 2020.
		negative control data	positive control data
	mean number of micronucleated cells per 4000 cells	3.6	44.6
	Standard deviation	1.4	29.9
	number of obsevations	41	38
	lower control limit (95% control limits)	1	-14
	upper control limit (95% control limits)	6	103

 

Conclusions:

Diammmonium hexachloroplatinate did not cause an increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of rats administered up to 150 mg/kg bw/day by gavage on three consecutive days. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.

Executive summary:

The in vivo clastogenicity and aneugenicity of diammonium hexachloroplatinate, 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 37.5, 75 or 150 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 no increase in the number of micronucleated polychromatic erythrocytes in any treatment group. On that basis, diammonium hexachloroplatinate was concluded to be non-genotoxic under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

In an OECD Test Guideline 471 study, conducted according to GLP, hexachloroplatinum(IV) solution was assessed for its ability to induce gene mutations in strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli (WP2 uvrA). The test was performed in two independent experiments, with dose levels determined following an initial toxicity test, both in the absence and presence of metabolic activation (S9) using liver fraction from rats pre-treated with phenobarbitone and beta-naphthoflavone. In experiment 1, dose-related increases in revertant numbers, which were at least two-fold the control values, were observed in WP2 uvrA both with and without S9. Dose-related increases in revertant numbers were also observed in TA98 and TA100 tester strains in the presence of S9. In experiment 2, conducted using the four S. typhimurium strains, large dose-related increases in revertant numbers were observed at higher dose levels in TA98 in the presence of S9. Dose-related and reproducible increases in revertant numbers were also observed in TA100 tester strain in the presence of S9. Although these increases did not reach two-fold the control values they were considered clear evidence of a mutagenic effect. It was concluded that hexachloroplatinum(IV) solution was mutagenic in S. typhimurium strains TA98 and TA100 and in E. coli WP2 uvrA under the experimental conditions (Scarcella, 2004).

 

The mutagenic potential of dihydrogen hexachloroplatinate (IV) solution was assessed in a reverse mutation assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assessed in four S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and in E. coli WP2 uvrA, in an attempt to detect both base-pair substitution and frameshift mutations. The test was performed in two independent experiments in the presence and absence of rat liver S9. In a dose range finding test, dihydrogen hexachloroplatinate (IV) solution was tested up to 5000 μg/plate in the absence and presence of 5% S9 mix with strains TA100 and WP2urvA. Based on the results of this test, in experiment 1 the test substance was tested up to 33 and 100 μg/plate in the absence and presence of 5% S9 mix, respectively. In experiment 2 the test substance was tested up to 33 and 100 μg/plate with and without 10% S9 mix, respectively. Toxicity was observed in all tested strains. In strain TA100, 1.8- and 1.7-fold increases in the number of revertant colonies were observed in the absence of S9 in the first and second experiments, respectively. These increases were not dose-related, were observed at the highest cytotoxic dose level only, and were not apparent in the dose-range finding study. However, since the increases were observed in two independently repeated experiments and outside the range of historical control data, these increases were considered by the investigators to be equivocal. A 2.3-fold increase in the number of revertant colonies was observed in the presence of S9 in the dose range finding study in strain TA100, though this result was not verified in experiments 1 and 2. However, since this mutagenic effect was more than two-fold and outside historical control data range, it was also considered to be equivocal. All other bacterial strains showed a negative response over the entire dose range in both experiments. It was concluded that dihydrogen hexachloroplatinate (IV) solution was equivocally mutagenic in the S. typhimurium strain TA100, and not mutagenic in E. coli (Verspeek-Rip, 2002).

 

In a limited published Ames test, dihydrogen hexachloroplatinate hexahydrate was weakly mutagenic according to the investigators in S. typhimurium strain TA98 when tested at up to 100 nmol/plate in the presence of S9 liver metabolic activation. It was negative in TA100 with S9, and in both strains without S9 (Uno and Morita, 1993).

 

However, in a limited Ames spot test, hexachloroplatinic acid failed to induce mutations in strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA1538) and E. coli (B/r WP2 try- and WP2 hcr- try-) when tested at up to 10 mM in the absence of metabolic activation (Kanematsu et al., 1980).

 

The ability of dihydrogen hexachloroplatinate(IV) solution to induce forward mutations at the thymidine-kinase (TK) locus in mouse lymphoma (L5178Y) cells was evaluated in an OECD Test Guideline 476 study, conducted according to GLP, both in the presence and absence of Aroclor-1254 induced rat liver S9-mix. A range-finding study was conducted to set dose levels for the subsequent mutation assay. In the main experiment, dihydrogen hexachloroplatinate(IV) solution was tested up to concentrations of 75 and 133 μg/mL, with an incubation time of 3 hours, with and without S9, respectively. The test material induced clear, dose-related, increases in the mutant frequency at the TK locus in the absence and presence of S9 (respective increases of 28.5- and 11.9-fold at the highest concentrations). The cell survival (compared to the solvent control) was reduced by 95% and 83% at the highest tested concentrations in the absence and presence of S9 -mix, respectively. Since a clear, dose dependent, positive response was observed in the mutant frequency at the TK locus after treatment, no repeat assay was performed. It was concluded that dihydrogen hexachloroplatinate(IV) solution was mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions (Verspeek-Rip, 2003).

 

In a limited bacterial rec assay with hexachloroplatinic acid, the observed difference in inhibition of bacterial growth was described by the investigators as a strong positive rec effect, indicating possible damage DNA (Kanematsu et al., 1980).

 

In a combined rat micronucleus test and comet assay following OECD 474 and 489, there was no increase in the number of micronucleated polychromatic erythrocytes and no increase in % tail intensity in liver, kidney, glandular stomach or duodenum in rats given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days (Eurlings, 2020). 

 

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 a range of in vitro studies (HCN, 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 (liability of ligands, number of active sites etc.) it is reasonable to expect that not all forms of platinum are carcinogenic (HCN, 2008). Limited experimental data on carcinogenicity for other platinum compounds give no evidence of activity that would meet classification criteria (HCN, 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 new, high-quality in vivo data showed diammonium hexachloroplatinate -and via read-across also hexachloroplatinic acid- itself to be conclusively non -genotoxic.

 

References

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

HCN (2008). Health Council of the Netherlands (DECOS). Platinum and platinum compounds. Health based recommended occupational exposure limit. https://www.gezondheidsraad.nl/sites/default/files/200812OSH_1.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, hexachloroplatinic acid does not currently meet the criteria for classification as a germ cell mutagen (category 1A/1B or 2) under EU CLP criteria (EC 1272/2008).