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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to OECD guidelines, with certain deviations.
Remarks:
Lacks a strain capable of detecting cross-linking mutagens, only tested at up to 1 mg/plate (instead of the recommended 5 mg/plate) and only plated in duplicate rather than in triplicate.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Guideline recommends the use of a strain capable of detecting cross-linking mutagens, which was not done. Only tested at up to 1 mg/plate (instead of the recommended 5 mg/plate). Only plated in duplicate rather than in triplicate.
Principles of method if other than guideline:
Method carried out according to Ames et al. (1975). Mutation Research 31, 347-364.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Metabolic activation system:
Rat liver microsomal fraction (S9) from male, CD1 rats induced with Aroclor 1254
Test concentrations with justification for top dose:
1.6, 8.0, 40, 200 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water (uncoded batch); isotonic saline (batch 031099/A)
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N- nitrosoguanidine
Remarks:
10 µg/plate for TA1535 and TA100 without S9
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
20 µg/plate for TA1537, TA 98 and TA100, with and without S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 µg/plate for TA1537 without S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
20 µg/plate for TA1538 and TA98 without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hr

OTHER: Plates prepared in duplicate.
Only tested up to 1 mg/ml although no problem with solubility was reported.
Tests with TA1537, TA98 and TA100 were conducted twice, both with and without S9 using the “uncoded” test compound in water. Three separate tests were performed with batch 031099/A dissolved in isotonic saline and TA1535, TA1537 and TA1538, only without S9.
Evaluation criteria:
No data, but reported the method to be according to Ames et al. (Mutation Research 1975, 31, 347-364), who considered a less than 2-fold increase in revertants, compared to spontaneous revertants, to be a negative response.
Statistics:
No data
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
other: weak positive
Cytotoxicity / choice of top concentrations:
other: cytotoxic at 1000 μg/plate in one of the duplicate tests, both in the presence and absence of S9
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
other: weak positive
Cytotoxicity / choice of top concentrations:
other: cytotoxic at 1000 μg/plate in one of the duplicate tests, both in the presence and absence of S9
Vehicle controls validity:
valid
Positive controls validity:
valid

TEST-SPECIFIC CONFOUNDING FACTORS

No data

 

RANGE-FINDING/SCREENING STUDIES: no data

 

COMPARISON WITH HISTORICAL CONTROL DATA: no data in the report [but revertants within the ranges given in the literature].

 

ADDITIONAL INFORMATION ON CYTOTOXICITY: in one of the duplicate tests there was a thinning of the background lawn reported for TA98 and TA100 together with a decrease in the number of revertants, both with and without S9. This was not evident with TA1537, or for TA 1535 and TA 1538 which were only tested without S9.

 

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

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

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

 

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

 

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Negative results were apparent in three in vivo studies on the test compound (a sex-linked recessive lethal test in Drosophila melanogaster, a micronucleus test in mice and a chromosome aberration test in Chinese hamsters) (Bootman and Lodge, 1980b; Bootman and Rees, 1981; Bootman and Whalley, 1980) and a further in vivo study on the structurally-related compound tetraammineplatinum hydrogen carbonate (an unscheduled DNA synthesis assay in rats) (Durward, 1999).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 September 1980 to 10 November 1980
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: See 'Remarks'
Remarks:
This study design does not comply with recent recommendations; at least 200 metaphases/animal should be scored for chromosome aberrations in order to provide sufficient statistical power. In addition it is recommended to administer the test material as a single dose since chromosomally damaged cells can die before the end of treatment with repeated dosing.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
Dosed on 5 consecutive days, only 1000 metaphases (at most) were scored
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
hamster, Chinese
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shell Research Laboratories
Sittingbourne
Kent
UK
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 27-33 g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: polypropylene cages
- Diet (e.g. ad libitum): sterile, conventional, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:
oral: gavage
Vehicle:

Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 4, 20 or 100 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Type and concentration of dispersant aid (if powder): gum tragacanth
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: freshly prepared daily prior to dosing
Duration of treatment / exposure:
5 days
Frequency of treatment:
Once daily
Post exposure period:
One day
Remarks:
Doses / Concentrations:
40, 200 or 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
chlorambucil
- Justification for choice of positive control(s): no data
- Route of administration: oral gavage
- Doses / concentrations: 15 mg/kg bw/day for 2 days

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on range-finding study in which groups of two male treated with 250-2000 mg/kg bw/day for 5 days and mitotic index calculated as an indication of toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treated for 5 days and sampled the following day. Vinblastine sulphate (spindle inhibitor) given by i.p. injection 2 hrs before sampling the bone marrow cells.

DETAILS OF SLIDE PREPARATION: Cells flushed out of the femurs, washed and resuspended in 0.075 M potassium chloride with heparin for 10 min, fixed in methanol : acetic acid (3:1) overnight before spreading on three slides per dose and air-drying. Stained with Giemsa and permanent mounts made.

METHOD OF ANALYSIS: slides randomly coded and 100 metaphases per dose scored for chromosome number and number and types of aberrations. Scoring was according to the ad hoc
Committee of the Environmental mutagen Society and the Institute for Medical Research (Chromosome methodologies in mutation testing, 1972. Toxicol. Appl. Pharmacol., 22, 269 – 275)

OTHER:

Evaluation criteria:
Only metaphases containing 22 ± 2 chromosomes were included in the total score.
Statistics:
Modified chi-squared calculation used to compare treated and control groups
Sex:
male
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
- Dose range: 250-2000 mg/kg bw/day for 5 days
- Solubility: given as a suspension
- Clinical signs of toxicity in test animals: weight loss, two animals of the top dose and one animal on 500 mg/kg bw/day died, or were humanely killed after three doses of the test substance.
- Evidence of cytotoxicity in tissue analyzed: dose-related decrease in mitotic index
- Rationale for exposure:
- Harvest times: day 6 (24 hr after the last treatment)


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): see table 1
- Statistical evaluation: no treated group differed significantly from the vehicle control group (P > 0.05)

 

Table 1. Comparison of aberrations found in the treated and vehicle control groups

 

Type of aberration

Total number of aberrations recorded

 

 

Group 1

(control)

Group 2

(40 mg/kgbw/day)

Group 3

(200 mg/kgbw/day)

Group 4

(1000 mg/kgbw/day)

Single-strand gaps

9

17

11

6*

Double-strand gaps

4

6

7

6*

Single-strand breaks

1

6

6

7*

Chromosome breaks

8

3

1

0*

Exchange figures

0

1

3

0*

Fragments

2

2

6

1*

Other aberrations

0

1

2

0*

                                                                  

* based on chromosome preparations from 4 animals

 

 

Conclusions:
Interpretation of results (migrated information): negative
Tetraammine platinum (II) chloride hydrate did not induce chromosome aberrations in bone marrow cells when Chinese hamsters were treated at up to 1000 mg/kg bw daily for 5 days by oral gavage.
Executive summary:
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.

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

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

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

 

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

 

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

 

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

 

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

 

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

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

 

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

 

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

 

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

 

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

 

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

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

 

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

 

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 for tetraammineplatinum dichloride itself. 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. the sex-linked recessive lethal test in D. melanogaster (OECD TG 477) and a liver unscheduled DNA synthesis assay (OECD TG 486, performed with tetraammineplatinum hydrogen carbonate)). Indeed, further 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 dichloride does not currently meet the criteria for classification as a germ cell mutagen (category 1A or 1B). However, this conclusion should be revisited when the results of the planned in vivo studies are available.