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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ammonium hexachloroplatinate was mutagenic in a bacterial reverse mutation (Ames) assay using four Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) when tested in the presence and absence of S9 (Bunger et al., 1996).

 

However, in a previously conducted Ames assay, ammonium hexachloroplatinate displayed no evidence of mutagenicity in S. typhimurium strains TA100, TA1535, TA1537 or TA1538 when tested at up to cytotoxic levels in the presence (TA100 and TA1537 only) or absence (all four strains) of S9. An inconsistent, weak positive result was seen in S. typhimurium TA98 in the presence, but not in the absence, of S9 (Bootman and May, 1980).

 

In a well-conducted in vitro mammalian gene mutation test using CHO cells, dipotassium hexachloroplatinate was weakly mutagenic when tested up to cytotoxic concentrations, in the absence of metabolic activation (Taylor et al., 1979).

 

Dipotassium hexachloroplatinate did not induce micronuclei in the cytokinesis-block micronucleus test with human lymphocytes, when tested at up to cytotoxic concentrations (Gebel et al., 1997).

 

However, according to a brief abstract, diammonium hexachloroplatinate was said to have shown a clear mutagenic effect, as indicated by the induction of a statistically higher number of micronuclei (MN) in two male donors than in controls in the dose-ranges of 75 -125 μM Pt. FISH analysis did not show a significant increase of MN-C (as a percentage), suggesting that the metal acts with both clastogenic and aneuploidogenic mechanisms (Migliore et al., 1999).

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:
weight of evidence
Study period:
not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to OECD guidelines. However, only four bacterial strains were tested (TA 1535 omitted).
Qualifier:
according to
Guideline:
other: Revised test protocol of Maron and Ames (1983)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The study differed principally from OECD TG471 in that only four bacterial strains were tested. The recommended strain TA1535 was ommitted.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidene operon
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
Sprague-Dawley rat liver, induced with Phenobarbital and beta-naphthoflavone
Test concentrations with justification for top dose:
The test substance was dissolved in distilled water and diluted to 5-500 ug/plate [or possibly 10, 50, 100 or 500 ug/plate] in all four tester strains, in the absence or presence of (4% and 10%) S9. The number of revertant colonies on the plates were recorded after 48 hours of incubation in the dark at 37degC.
Vehicle / solvent:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Details on test system and experimental conditions:
The tests were done in duplicate and repeated at least 3 times.
Evaluation criteria:
For the test substance to be considered mutagenic, a two-fold (or more) increase in the mean revertant numbers must be observed in the plates containing the test substance compared to the spontaneous reversion rate.
Statistics:
None used
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100 and TA102
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100 and TA102
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Additional information on results:
The test substance caused a 2- to 10-fold increase in revertants in all four tester strains (compared with spontaneous reversion rates), in the presence and absence of S9.
"The increase in reverse mutation rates in the samples that tested positive was dosage-dependent".

"High doses of the metal compounds proved toxic to the tester strains", resulting in a thinning of the background bacterial lawn. Although no actual data were provided for ammonium hexachloroplatinate, the minimum toxic dose for the platinum salts was apparently 100 ug/plate.

Conclusions:
Interpretation of results (migrated information):
positive

Ammonium hexachloroplatinate was mutagenic in a bacterial reverse mutation (Ames) assay using four Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) when tested in the presence and absence of S9.
Executive summary:

Ammonium hexachloroplatinate was assessed for mutagenic activity in a bacterial reverse mutation (Ames) assay, similar to OECD Test Guideline 471, using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 and tested at up to 500 μg/plate in the presence and absence of a metabolic activation system (S9) derived from phenobarbital- and beta-naphthoflavone-induced rat livers. (It is not clear whether the di- or mono-ammonium form was used, see "Test materials" section; the recommended strain TA1535 was omitted.)

 

Mutagenic effects were seen in all four strains both in the presence and absence of metabolic activation. Although no cytotoxicity data were provided for the test material, the minimum toxic dose for the platinum salts was apparently 100 μg/plate.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data were identified.

 

The observation of mutagenic activity in bacterial (Bootman and May, 1980; Bunger et al., 1996; Kanematsu et al., 1980) and mammalian cells (Taylor et al., 1979) as well as cytogenicity in mammalian cells (Migliore et al., 1999) necessitates the consideration of further in vivo testing. Related platinum compounds have also demonstrated a general tendency to induce genotoxicity in vitro, though the available, if somewhat limited, dataset suggests that such findings might not be relevant in vivo and hence a similar lack of classification for mutagenicity is proposed for these compounds. Additional in vivo testing of certain related platinates has been proposed to further elucidate the in vivo relevance of the in vitro findings. The conclusion not to classify diammonium hexachloroplatinate as a germ cell mutagen should be revisited when the results of the planned in vivo studies (see testing proposals) are available.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

Ammonium hexachloroplatinate was assessed for mutagenic activity in a bacterial reverse mutation

(Ames) assay, similar to OECD Test Guideline 471, using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 and tested at up to 500 μg/plate in the presence and absence of a metabolic activation system (S9) derived from phenobarbital- and beta-naphthoflavone-induced rat livers. (It is not clear whether the di- or mono-ammonium form was used; the recommended strain TA1535 was omitted.) Mutagenic effects were seen in all four strains both in the presence and absence of metabolic activation. Although no cytotoxicity data were provided for the test material, the minimum toxic dose for the platinum salts was apparently 100 μg/plate (Bunger et al., 1996). This study conforms to OECD guidelines, aside from the lack of inclusion of a fifth tester strain.

 

In a previously conducted study, the mutagenic activity of ammonium hexachloroplatinate in S. typhimurium was investigated in an Ames assay, similar to OECD Test Guideline 471, using pour-plate assays. The bacterial strains employed are capable of detecting both induced frameshift (TA1537, TA1538, and TA98) and base-pair substitution (TA1535 and TA100) mutations. Tests with strains TA98, TA100 and TA1537 were conducted at least in duplicate with the test material dissolved in deionised water at up to 1000 μg/plate (and in a subsequent test, due to inhibition of growth seen at the higher levels, at up to 100 μg/plate), both with and without S9. Two further tests (both in duplicate) were conducted in all five strains in the absence of S9, with the test material dissolved in isotonic saline solution at up to 100 μg/plate. No evidence of mutagenic activity was seen in any test with strains TA1535, TA1537, TA1538 or TA100, at up to cytotoxic levels, in the absence or presence of S9 (when used). A doubling of revertant colonies (weak positive) was seen in strain TA98 using the test material in deionised water at 40 μg/plate (test 1), and 4 and 20 μg/plate (test 2), in the presence of S9, but not in its absence. A further test with TA98 (test 3), again at up to 100 μg/plate (cytotoxic level), found no doubling in revertant colonies compared to spontaneous vehicle controls, with or without S9. Therefore, the study authors concluded that “no consistent evidence was obtained of mutagenic activity with strain TA98” (Bootman and May, 1980). This study does not entirely conform to current OECD guidelines which recommend including a bacterial strain which is susceptible to oxidative mutagenesis or cross-linking agents, for example S. typhimurium TA102, or Escherichia coli WP2 uvrA. Also, where the test material was dissolved in saline, no S9 was used, which is contrary to the OECD guidelines that recommend exposing bacteria to the test substance in the presence and absence of an appropriate metabolic activation system.

 

In a limited Ames spot test, diammonium hexachloroplatinate induced reverse mutations in S. typhimurium strain TA98 and E. coli strain WP2 hcr- try- when tested at up to 10 mM in the absence of metabolic activation (Kanematsu et al., 1980).

 

Dipotassium hexachloroplatinate was tested for genotoxicity in an in vitro mammalian gene mutation test using Chinese Hamster Ovary (CHO) cells deficient in HPRT (hypoxanthine-guanine phosphoribosyl transferase). Cells were tested only in the absence of a metabolic activation system. A 2-3-fold increase in the 8-AGR mutant frequency versus the spontaneous control was seen upon repeated subculture [prolonged exposure] with a non-cytotoxic concentration of 10 μM. This increase was apparent after 10 population doublings; the trend towards it was observed at 5 population doublings. Dipotassium hexachloroplatinate was determined to be weakly mutagenic to mammalian cells in the absence of metabolic activation under the conditions of the test (Taylor et al., 1979).

 

In a well-conducted study, similar to that described by OECD Test Guideline 487, the ability of dipotassium hexachloroplatinate to induce micronuclei in human peripheral mononuclear blood cells (lymphocytes) was assessed, in the absence of a metabolic activation system. The mean numbers of micronuclei in binucleate cells were 4, 5.5, 7.5 and 4 at test concentrations of 0, 5, 10 and 20 µM, respectively; there was no statistically significant change compared to the negative control. Severe cytotoxicity was observed at 40 µM. In conclusion, the test substance did not cause chromosome damage (micronuclei) in the cytokinesis-block micronucleus test with human lymphocytes (Gebel et al., 1997).

 

However, a brief abstract presents details of an in vitro micronucleus test using human lymphocytes from the whole blood of two young, non-smoking, males. Cells were blocked in cytokinesis by cytochalasin B. Micronuclei (MN) frequency was evaluated in binucleated lymphocytes and MN were analysed for the presence of a fluorescent signal by considering a labelled MN as centromere-positive MN (C+MN). Mytomycin (MMC, clastogen) and griseofulvin (GF, aneuploidogen) were used as positive standard mutagens. In addition, the fluorescence in situ hybridisation (FISH) technique with an alphoid centromere-specific DNA probe was applied in order to clarify the mechanism of action. Diammonium hexachloroplatinate was said to have shown a clear mutagenic effect, as indicated by the induction of a statistically higher number of MN in both donors than in controls in the dose-ranges of 75 -125 uM Pt. FISH analysis did not show a significant increase of MN-C (as a percentage), suggesting that the metal acts with both clastogenic and aneuploidogenic mechanisms (Migliore et al., 1999).

 

Dipotassium hexachloroplatinate did not cause DNA damage in a bacterial SOS chromotest, when tested at up to cytotoxic concentrations in the absence of metabolic activation (Gebel et al., 1997; Lantzsch and Gebel, 1997).

 

In a limited bacterial rec assay with ammonium hexachloroplatinate, 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).

 

No in vivo genotoxicity data were identified.

Dipotassium hexachloroplatinate is considered to fall within the scope of the read-across category "hexachloroplatinate(IV) compounds". 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 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 available in vivo data returned mostly negative results. 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 dipotassium tetrachloroplatinate) 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

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, diammonium hexachloroplatinate does not currently meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008). However, this conclusion should be revisited when the results of the planned in vivo studies are available.