Registration Dossier
Registration Dossier
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EC number: 848-537-7 | CAS number: 1912392-88-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In a bacterial reverse mutation (Ames) test, conducted according to OECD Test Guideline 471 and to GLP, di(tetraethylammonium)hexahydroxoplatinate induced reverse mutations in strains of Salmonella typhimurium and Escherichia coli both in the absence and presence of a rat liver metabolic activation system (S9) (Thompson, 2019).
No in vitro mammalian cell cytogenicity/mutagenicity data are available for di(tetraethylammonium)hexahydroxoplatinate. Further in vitro testing is considered unnecessary in the light of the performed in vivo genotoxicity testing.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
- Test concentrations with justification for top dose:
- Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000, 1812 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 1000, 1812 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 1, 3, 10, 33, 100, 333, 1000, 1812 µg/mL
With S9-mix, 3 hours treatment: 1, 3, 10, 33, 100, 333, 1000, 1812 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1, 3, 10, 33, 100, 333, 1000, 1812 µg/mL
With S9-mix, 3 hours treatment: 1, 3, 10, 33, 100, 333, 1000, 1812 μg/mL - Vehicle / solvent:
- - Solvent used: RPMI 1640 (Exposure medium, Hepes buffered medium (Dutch modification) (Invitrogen Corporation, Breda, The Netherlands)).
- Justification for choice of solvent/vehicle:
Test compound was soluble in RPMI 1640 and RPMI 1640 has been accepted and approved by authorities and international guidelines. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- other: 15 and 5 μg/ml for 3 and 24 hours treatment period, respectively.
- Remarks:
- without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: 10 μg/ml.
- Remarks:
- with S9: 7.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures
NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments) - Evaluation criteria:
- ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (106) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 106 survivors, and for CP not below 700 per 106 survivors.
DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test. - Statistics:
- The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No, pH at highest concentration (1812 μg/ml (= 10 mM)) was 7.9 versus 7.3 in the solvent control.
- Effects of osmolality: No, osmolality at highest concentration (1812 μg/ml (= 10 mM)) was 0.308 Osm/kg versus 0.311 Osm/kg in the solvent control.
- Precipitation: No precipitation in the exposure medium was observed at any concentration.
RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to the precipitating dose levels of 1812 μg/ml in the absence and presence of S9-mix.
COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested dose level in both experiments in the absence and presence of S9-mix. - Remarks on result:
- other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
- Conclusions:
- Based on a mouse lymphoma assay conducted according to OECD 476 guideline and GLP principles, it can be concluded that TMAH is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
- Executive summary:
A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, TMAH did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of 8% v/v S9-mix, TMAH did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with 12% v/v S9 for metabolic activation. It is concluded that TMAH is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
- 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:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3341/00-F9
- Expiration date of the lot/batch: September 10, 2020
- Purity test date: Certificate of analysis dated September 09, 2019
- Purity: 31.3 % w/w
- Form: liquid
- Appearance: amber liquid - 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:
- Sprague-Dawley rat liver induced with phenobarbital-5,6 and benzoflavone
- Test concentrations with justification for top dose:
- In the plate incorporation method test the maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. - Vehicle / solvent:
- Sterile distilled water
- Negative solvent / vehicle controls:
- yes
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the mutation test employing the plate incorporation method.
A test item precipitate (white and particulate in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in the mutation test. This observation did not prevent the scoring of revertant colonies.
In the absence of metabolic activation, statistically significant increases in TA100 revertant colony frequency were noted from 50 μg/plate, although this particular response was quite flat over the statistically significant concentrations. However, dose-related and statistically significant increases were noted for WP2uvrA from 15 μg/plate and TA98 from 1500 μg/plate. The increases observed for WP2uvrA were particularly large and in excess of the upper in-house historical control maxima range with a maximum fold increase of 7.1 fold over the concurrent vehicle control noted at 5000 μg/plate. A 2.4 fold increase over the concurrent vehicle control was noted for TA98 at 5000 μg/plate.
Larger increases were noted in the presence of metabolic activation with dose-related and statistically significant increases noted from 150 μg/plate for TA100 and TA98 and
50 μg/plate for WP2uvrA. The increases observed for each strain were large and in excess of the upper in-house historical control maxima range with maximum fold increases over the concurrent vehicle controls noted of 2.3 for TA100, 7.2 for WP2uvrA and 3.8 for TA98.
Any excursions outside the maxima ranges, particularly when a dose-response relationship are apparent, must be considered to be evidence of a biological response.
No increases in colony frequency were noted at any test item concentration for bacterial strains TA1535 and TA1537 dosed in either the absence or presence of metabolic activation (S9-mix). - Conclusions:
- In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item di(tetraethylammonium)hexahydroxoplatinate induced substantial increases in the frequency of TA100, WP2uvrA and TA98 revertant colonies both with and without metabolic activation (S9-mix). Under the conditions of this test di(tetraethylammonium)hexahydroxoplatinate was considered to be mutagenic.
- Executive summary:
In an OECD Test Guideline 471 study, conducted according to GLP, di(tetraethylammonium)hexahydroxoplatinate was assessed for its ability to induce gene mutations in strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2 uvrA).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Reproducible, large and dose-related increases in the number of revertant colonies, which were more than two-fold the control values, with TA98, TA100 and WP2 uvrA tester strains in the plate incorporation assays, both in the presence and absence of S9 metabolism were observed. No increases in colony frequency were noted at any test item concentration for bacterial strains TA1535 and TA1537 dosed in either the absence or presence of metabolic activation.
No second experiment, including a pre-incubation step, was performed as the OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment.
It was concluded that di(tetraethylammonium)hexahydroxoplatinate was mutagenic in S. typhimurium and E.coli under the reported experimental conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted according to GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- 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:
- Sprague-Dawley rat liver induced with phenobarbitone and betanaphthoflavone
- Test concentrations with justification for top dose:
- Two main experiments.
In the first, using the plate incorporation method, the test substance was assayed at 313, 625, 1250, 2500 or 5000 ug/plate in all five tester strains, in the absence or presence of S9. In the second, TA98, TA100, and WP2 uvrA strains were tested under the same experimental conditions as the first experiment, while for TA1535 and TA1537, a pre-incubation step was included. - Vehicle / solvent:
- Sterile distilled water
- Untreated negative controls:
- yes
- Remarks:
- Dimethoxysulphoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Untreated
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Migrated to IUCLID6: in distilled water
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Migrated to IUCLID6: in DMSO
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Migrated to IUCLID6: in DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene in DMSO
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Migrated to IUCLID6: in distilled water
- Evaluation criteria:
- For the test substance to be considered mutagenic, a two-fold (or more) increase in the mean revertant numbers must be observed at two-consecutive dose-levels or at the highest practiable dose-level only. In addition, there must be evidence of a dose-reponse relationship.
- Statistics:
- Regression analysis by the least squares method
- Species / strain:
- S. typhimurium, other: TA98, TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In main experiment one, two-fold, dose-related increases in revertant colonies compared to controls were seen in TA98, TA100, and WP2 uvrA strains, with and without S9.
In main experiment two, the test substance induced reproducible, dose-related, two-fold increases in the number of revertant colonies compared to controls in TA98, TA100, and WP2 uvrA strains, with and without S9. Two-fold increases in TA1537 were also seen using the pre-incubation method, at non-toxic dose-levels, with and without S9. - Conclusions:
- In an OECD Test Guideline 471 study, to GLP, dihydrogen hexachloroplatinate, compound with 2-aminoethanol (1:2) induced reverse mutations in strains of Salmonella typhimurium and Escherichia coli both with and without metabolic activation.
- Executive summary:
In an OECD Test Guideline 471 study, conducted according to GLP, dihydrogen hexachloroplatinate, compound with 2-aminoethanol (1:2) was assessed for its ability to induce gene mutations in strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2 uvrA). The test was performed in two independent experiments (involving the plate incorporation method, including a pre-incubation step for TA1535 and TA1537 in Experiment 2), with dose levels of up to 5000 μg/plate (determined following an initial toxicity test), both in the absence and presence of rat liver metabolic activation (S9).
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. The test item induced reproducible, large and dose-related increases in the number of revertant colonies, which were more than two-fold the control values, with TA98, TA100 and WP2 uvrA tester strains in the plate incorporation assays, at the higher dose levels both in the presence and absence of S9 metabolism. Increases in revertant numbers were also observed with TA1537 in the pre-incubation assay. These increases were greater than twice the control value at non-toxic dose-levels, both in the presence and absence of S9 metabolism.
It was concluded that dihydrogen hexachloroplatinate, compound with 2-aminoethanol (1:2) was mutagenic in S. typhimurium and E.coli under the reported experimental conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 5 December 2001 - 2 May 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted according to GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 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:
- HGPRT gene
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Sprague-Dawley rat liver induced with phenobarbitone and betanaphthoflavone
- Test concentrations with justification for top dose:
- Concentrations of 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 ug/ml were used in the absence of S9, and 313, 625, 1250, 2500 and 5000 ug/ml in the presence of S9.
- Vehicle / solvent:
- Culture medium
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- in ethanol served as positive control in absence of S9
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- in DMSO as positive control in the presence of S9
- Details on test system and experimental conditions:
- The cultures were incubated at 37 degrees C for three hours, before the treatment medium was removed and washed with phosphate buffered saline. Fresh complete medium was added to the culture which was returned to the incubator. On day six (first expression time) and day eight (second expression time), each culture was trypsinised, resuspended in complete medium and counted microscopically. After dilution, about 100,000 cells were plated and supplemented with 6-thioguanine (at 7.5 ug/ml), stained and scored for the presence of mutants.
- Evaluation criteria:
- Test substance considered mutagenic, if it induces a five-fold (or more) increase in mutation frequency compared with solvent controls, over two consecutive doses. If only the highest practicable dose-level gives such an increase, then a single treatment-level will suffice. A dose-relation must be evident.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see below
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the preliminary cytotoxicity test in the absence of S9, survival was reduced to below the limit of detection at concentrations between 625 and 5000 ug/ml. At 78.1, 156 and 313 ug/ml, severe toxicity was seen, while at the remaining concentrations (39.1 and 19.5 ug/ml) moderate toxicity was observed. In the presence of S9, moderate toxicity was seen at 5000 ug/ml (highest tested dose).
A greater than five-fold, dose-related increase in the frequency of spontaneous mutations were seen (in duplicate cultures) at both expression times compared to negative controls, in the presence or absence of S9. - Conclusions:
- In an in vitro mammalian cell gene mutation assay, conducted in accordance with OECD Test Guideline 476 and to GLP, dihydrogen hexachloroplatinate, compound with 2-aminoethanol (1:2) induced mutations in Chinese hamster V79 cells when tested both with and without S9.
- Executive summary:
In an in vitro GLP study, conducted in accordance with OECD Test Guideline 476 (in vitro mammalian cell gene mutation assay), dihydrogen hexachloroplatinate, compound with 2-aminoethanol (1:2) was tested for its ability to induce 6 -thioguanine resistant mutants in Chinese hamster V79 cells.
Cells were exposed to test material for 3 hours, both in the presence and absence of metabolic activation using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. On the basis of a preliminary cytotoxicity test, the maximum dose levels for the mutation assay were selected as 78.1 and 5000 μg/ml for treatment in the absence or presence of S9, respectively.
Dose related and significant increases in mutant numbers or mutant frequency were observed following treatment with the test item, in the absence and presence of S9 metabolism. These increases were greater than five-fold the spontaneous mutation frequency, in replicate cultures at both expression times (days, 6 and 8). It was concluded that the test substance was mutagenic in Chinese hamster V79 cells, both in the presence and absence of S9.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 30 July to 2 September 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, performed to GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Minor deviations did not affect the overall interpretation of study findings or compromise integrity of the study
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine locus
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (main study (Experiments 1 and 2))
- Additional strain / cell type characteristics:
- other: histidine-requiring
- Species / strain / cell type:
- other: S. typhimurium TA 98, TA 100 and TA 102 (range-finding study)
- Additional strain / cell type characteristics:
- other: histidine-requiring
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S-9
- Test concentrations with justification for top dose:
- Range-finding study: 0, 5, 15.8, 50, 158.1, 500, 1581 or 5000 µg/plate
Main study:
Experiment 1: 0, 5, 15.8, 50, 158.1, 500, 1581 or 5000 µg/plate
Experiment 2: 0, 156.3, 312.5, 625, 1250, 2500 or 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that dihydrogen hexahydroxyplatinate was insoluble in several commonly used solvents including water, acetone, anhydrous analytical grade dimethyl sulphoxide (DMSO), ethanol, tetrahydrofuran and dimethylformamide (DMF). The test article formed a homogenous suspension at approximately 50 mg/mL in water and at approximately 299 mg/mL in DMSO, DMF and acetone. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 5 µg/plate
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA98, without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 2 µg/plate
- Positive control substance:
- sodium azide
- Remarks:
- TA100 and TA1535, without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 50 µg/plate
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537, without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.2 µg/plate
- Positive control substance:
- mitomycin C
- Remarks:
- TA102, without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/plate
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- TA98, with S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 5 µg/plate (TA100, TA1535, TA1537), 20 µg/plate (TA102)
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- TA100, TA102, TA1535 and TA1537, with S-9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 20 min (Experiment 2, TA1535 and TA1537 with S-9)
- Exposure duration: 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): ~3 days
NUMBER OF REPLICATIONS: single test plates (range-finding study); triplicate (main study (Experiments 1 and 2))
SELECTION AGENT (mutation assays): histidine-free medium
DETERMINATION OF CYTOTOXICITY
- Method: other: background lawns examined for signs of toxicity (e.g. marked reduction in revertants compared to controls) - Evaluation criteria:
- For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p≤0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example
consistency of response within and between concentrations and (where applicable) between experiments. - Statistics:
- Dunnett's test was used to assess the probability of the observed results arising by chance. Results were considered statistically significant when p≤0.01.
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 1 at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- ambiguous
- Remarks:
- statistically significant increase in revertants in Experiments 1 and 2, but not clearly concentration-related or reproducible
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- in Experiment 2, with pre-incubation
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 1 from 500 or 1581 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- ambiguous
- Remarks:
- statistically significant increase in revertants in Experiment 2, but not clearly concentration-related or reproducible
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 1 from 500 or 1581 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 2, at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- See tables 1 and 2 for revertant numbers/plate for experiments 1 and 2.
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: noted in all strains treated with 5000 µg/plate, with and without S-9 (in Experiments 1 and 2)
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: no evidence of cytotoxicity in tested strains (TA98, TA100 and TA102). Range-finding data were considered to be acceptable for cytotoxicity assessment only.
COMPARISON WITH HISTORICAL CONTROL DATA: results for vehicle controls were compared to historical control data from within the laboratory.
ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was observed as a slight thinning of the background bacterial lawn, a marked reduction in revertant numbers, or a complete killing of the test bacteria. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive
In a good-quality Ames assay, conducted according to GLP and OECD Test Guideline 471, dihydrogen hexahydroxyplatinate displayed evidence of mutagenicity when tested in five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537), in the presence and absence of a rat liver metabolic activation (S9) system. - Executive summary:
Dihydrogen hexahydroxyplatinate was assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Guideline 471. Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were tested with and without the addition of a mammalian (rat liver) metabolic activation (S9) system in two separate experiments.
In the first experiment, agar containing the test substance at up to 5000 µg/plate was incubated with the bacterial strains for 3 days. The second experiment, also using concentrations of up to 5000 µg/plate, included an additional 20-minute pre-incubation step for cultures of TA1535 and TA1537 in the presence of S9.
There was significant evidence of mutagenicity in strains TA98 and TA100, with and without S9, and TA1535 and TA102 in the presence of S9 only. The lowest concentration producing a statistically significant increase in the number of revertant colonies was 50 µg/plate (for TA100, with S9). Cytotoxicity was observed at high concentrations in some of the test plates.Vehicle and positive controls performed as expected.
Under the conditions of this assay, dihydrogen hexahydroxyplatinate was mutagenic to S. typhimurium, with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1 August to 24 October 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, performed to GLP.
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: McCoy's 5A medium including 10% (v/v) heat inactivated foetal calf serum (FCS), and 0.52% Penicillin/Streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S-9
- Test concentrations with justification for top dose:
- Range-finding study 1: 0 or 0.9-250 µg/ml
Range-finding study 2: 0 or 10.1-1000 µg/ml
Main study: 0 or 10-800 µg/ml
Concentrations assessed in main study:
3-hr (with and without S-9): 0, 20, 40, 70 or 100 µg/ml
24-hr (without S-9): 0, 10, 20, 40 or 70 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 1% w/v methyl cellulose
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that dihydrogen hexahydroxyplatinate was insoluble in water, acetone, dimethyl sulphoxide, ethanol, tetrahydrofuran and dimethylformamide - Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- methyl cellulose
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without S-9 (3-hr treatment), at 0.06, 0.08 or 0.12 µg/ml
- Positive control substance:
- mitomycin C
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- methyl cellulose
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with S-9 (3-hr treatment), at 6 or 8 µg/ml
- Positive control substance:
- cyclophosphamide
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- methyl cellulose
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without S-9 (24-hr treatment), at 0.003 or 0.005 µg/ml
- Positive control substance:
- other: vinblastine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 0 hours
- Exposure duration: 3 (with and without S-9) or 24 hours (without S-9)
- Expression time (cells in growth medium): 21 (with and without S-9) or 0 hours (without S-9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline (PBS)
NUMBER OF REPLICATIONS: duplicate
NUMBER OF CELLS EVALUATED: range-finding study: at least 200/concentration; main study: 1000/culture (2000/concentration)
DETERMINATION OF CYTOTOXICITY
- Method: other: replication index
OTHER EXAMINATIONS:
- Determination of polyploidy: no data
- Determination of endoreplication: no data
- Other: no data - Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of micronucleated binucleate (MNBN) cells at one or more concentrations was observed
2. An incidence of cells with micronuclei at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of cells with micronuclei was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations. - Statistics:
- After completion of scoring and decoding of slides, the numbers of MNBN cells in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportions of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test.
Probability values of p≤0.05 were accepted as significant. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- ambiguous
- Remarks:
- frequency of micronuclei statistically significantly higher than control, but within normal range except one culture at 70 µg/ml, for which MNBN cell incidence exceeded the normal range
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 9, 10, 19 and 26% for selected test concentrations (20, 40, 70 and 100 µg/ml, respectively). Higher levels of cytotoxicity seen at higher concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- significantly increased incidence of micronuclei seen at each test concentration
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 8, 19, 34 and 43% for selected test concentrations (20, 40, 70 and 100 µg/ml, respectively). Higher levels of cytotoxicity seen at higher concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- not determined
- Remarks:
- no significant increase in micronuclei relative to controls, but positive control not valid
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 6, 6, 12 and 12% for selected test concentrations (10, 20, 40 and 70 µg/ml, respectively). Higher levels of cytotoxicity seen at higher concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked change in pH were observed at any tested concentration
- Effects of osmolality: no marked changes seen in range-finding study at up to 1000 µg/ml
- Evaporation from medium: no data
- Water solubility: see "precipitation" below
- Precipitation: seen at various concentrations in the range-finding test. In the main study, precipitation was seen from the lowest concentration with and without S-9 (3-hour and 24-hour treatments).
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: range-finding studies for cytotoxicity used concentrations of up to 250 (study 1) or 1000 µg/ml (study 2). In the first range-finding study, the highest level of cytotoxicity was 34%, seen at 250 µg/ml without S-9 (3-hour treatment). In the second range-finding study, the highest level of cytotoxicity was 56%, seen at 1000 µg/ml without S-9 (3-hour treatment).
COMPARISON WITH HISTORICAL CONTROL DATA: vehicle data were compared to historical control data. See Table 1 in "Any other information on results incl. tables" for the relevant historical control ranges. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'. Remarks: 3-hour treatment
- Conclusions:
- Interpretation of results (migrated information):
positive
In an in vitro mammalian cell micronucleus test, conducted according to GLP and OECD Test Guideline 487, dihydrogen hexahydroxyplatinate induced micronuclei in cultured Chinese hamster ovary (CHO) cells following treatment with metabolic activation (S9). Weak evidence for inducing micronuclei was seen in the absence of S9. - Executive summary:
In an in vitro GLP study, conducted in accordance with OECD Test Guideline 487 (in vitro mammalian cell micronucleus test), dihydrogen hexahydroxyplatinate was tested for its ability to induce chromosome damage in the form of micronuclei.
Following two range-finding studies, Chinese hamster ovary (CHO) cells were treated with the test substance (in 1% methyl cellulose) for 3 hours (with a 21-hr recovery phase) in the presence and absence of rat liver (S9) metabolic activation, or for 24 hr in the absence of S9 alone. The maximum assessed concentrations, limited by precipitation of the test material, were 100 and 70 μg/ml for cells treated for 3 and 24 hr, respectively.
For the 3-hr treatment in the presence of S9, there was clear evidence of chromosome damage from the lowest tested concentration (20 µg/ml). In the absence of S9, results were ambiguous – statistically significantly higher incidences of micronuclei were observed relative to the vehicle control, but incidences were generally within the normal historical control range. The results from the 24-hour continuous treatment were not considered reliable; no significant genotoxicity was seen for the test substance or for the positive control. Nevertheless, results from the 3-hour treatment were considered sufficient to conclude that dihydrogen hexahydroxyplatinate is clastogenic under the conditions of this assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, performed to GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine locus
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- The liver microsome fraction (S9 mix) was prepared from the liver of Sprague-Dawley rats pretreated with phenobarbital and 5,6-benzoflavon.
- Test concentrations with justification for top dose:
- 39.1; 78.1; 156; 313; 625 and 1250 ug/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.5; 1; 2 or 10 ug/plate
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- with S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.5 µg/plate
- Positive control substance:
- sodium azide
- Remarks:
- without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 80 µg/plate
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.01 or 0.1 µg/plate
- Positive control substance:
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide
- Remarks:
- without S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/plate
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- TA98, with S-9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMF
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 5 µg/plate (TA100, TA1535, TA1537), 20 µg/plate (TA102)
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- TA100, TA102, TA1535 and TA1537, with S-9
- Details on test system and experimental conditions:
- The test was conducted by the preincubation method and triplicate plates were used for each of six different concentrations of the test substance. The liver microsome fraction (S9 mix) was prepared from the liver of Sprague-Dawley rats pretreated with phenobarbital and 5,6-benzoflavon.
- Evaluation criteria:
- The test substance was considered positive in mutagenicity if the number of revertants found was more than twice the number of those of the solvent (water) control and if reproducible or concentration-dependent increase in the number of the revertants were observed.
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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:
- negative
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- in Experiments 1 and 2
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- ambiguous
- Remarks:
- statistically significant increase in revertants in Experiments 1 and 2, but not clearly concentration-related or reproducible
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- ambiguous
- Remarks:
- statistically significant increase in revertants in Experiment 2, but not clearly concentration-related or reproducible
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 1 from 500 or 1581 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- in Experiment 2, with pre-incubation
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 1 from 500 or 1581 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment 2, at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- See tables 1 and 2 for revertant numbers/plate for experiments 1 and 2 (attached illustration).
- Remarks on result:
- other: Considerable growth inhibition was observed in all strains treated at the highest concentrations of tetramethylammonium hydroxide. However, no increase in the number of revertant colonies was observed in either strain (S. typhimurium TA98, TA100, TA1535,
- Conclusions:
- Interpretation of results:
negative
In a good-quality Ames assay, conducted according to GLP and OECD Test Guideline 471, tetramethylammoniumhydroxide did not show any mutagenic activity in any tester strains with or without metabolic activation (TA98, TA100, TA1535, TA1537 and WP2 uvrA). - Executive summary:
Tetramethylammoniumhydroxide was assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Guideline 471. Mutagenicity was examined in four Salmonella typhimurium strains, TA98, TA100, TA1535 and TA1537, and in an Escherichia coli strain, WP2 uvrA, at concentrations up to growth inhibition dose (1.25 mg/plate for S. typhimurium strains and 5 mg/plate for E. coli strain with exogenous metabolic activation system) with and without exogenous metabolic activation system.In this study, the substance did not show any mutagenic activity in any tester strains with or without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, performed to GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9
- Test concentrations with justification for top dose:
- 228; 455 and 910 ug/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without S-9
- Positive control substance:
- mitomycin C
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with S-9
- Positive control substance:
- cyclophosphamide
- Details on test system and experimental conditions:
- Duplicate plates were used for each of three different concentrations of
228, 455 and 910 ug/ml. Cells were treated continuously with the test substance for 24 hr in the absence of metabolic activation (continuous treatment) or shortly for 6 hr with the test substance (short-term treatment) in the presence or absence of metabolic activation. In the short-term treatment, cells were incubated for additional 18 hr in a fresh culture medium without the test substance. The co-factor-supplemented post- mitochondrial fraction (S9 mix) was prepared from the livers of male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavon. Mytomycin C or cyclophosphamide was used as a positive control for the assay.
Colcemid (0.2 ug/ml) was added to the culture medium 2 hr before cell harvesting and then chromosome preparations were made. A hundred well-spread metaphases were observed under a microscope for each plate. Incidence of polyploid cells and that of cells with structural aberrations such as chromatid breaks and exchanges, chromosome breaks, exchanges and gaps, and others were recorded. - Evaluation criteria:
- Colcemid (0.2 ug/ml) was added to the culture medium 2 hr before cell harvesting and then chromosome preparations were made. A hundred well-spread metaphases were observed under a microscope for each plate. Incidence of polyploid cells and that of cells with structural aberrations such as chromatid breaks and exchanges, chromosome breaks, exchanges and gaps, and others were recorded.
The results were considered negative when the incidence was less than 5%. - Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No considerable inhibition of cell growth was observed up to the highest dose (910 ug/ml), either by continuous or short-term treatment regardless of metabolic activation.
No increase in the number of cells with structural chromosomal aberrations with or without gaps was observed at any doses after the continuous treatment or the short-term treatment with or without metabolic activation while marked increase in the number of those cells was observed in cultures treated with mytomicin C (continuous treatment and short-term treatment without metabolic activation) or cyclophosphamide (short-term treatment with metabolic activation). No increase in the number of polyploid cells was observed either.
Based on these findings, tetramethylammonium hydroxide was considered negative in the induction of chromosomal aberrations. - Remarks on result:
- other: see tables 1, 2 and 3 (attached illustration)
- Conclusions:
- Interpretation of results:
negative
In a good-quality Chromosome aberration test, conducted according to GLP and OECD Test Guideline 473, tetramethylammoniumhydroxide did not show any mutagenic activity. - Executive summary:
Tetramethylammoniumhydroxide (TMAH) was assessed for mutagenicity in a Chromosome aberration test performed to GLP, and according to OECD Guideline 473. The CHL/IU cells were treated continuously (24 hours) in the absence of metabolic activation and shortly (6 hours) in the presence or absence of metabolic activation with TMAH at concentrations of 0.228, 0.455 and 0.910 mg/ml (limited concentration, 10mM). No increase in clastogenicity (structural chromosome aberration) or polyploidy was observed in either conditions although the pH of the culture media showed a slight increase in alkalinity at the highest concentration.
Referenceopen allclose all
Addition of test substance did not have any obvious effect on the osmolatiy or pH of the treatment medium.
Precipitation seen at 625, 1250, 2500 and 5000 ug/ml.
Table 1: Number of revertants per plate (mean of 3 plates (5 for negative control)) – Experiment 1
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Conc. |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
0 (DMF) |
22.8 |
24.0 |
96.2 |
89.4 |
235.0 |
198.8 |
21.2 |
16.6 |
8.2 |
11.0 |
5 |
16.3 |
27.3 |
72.0 |
84.7 |
242.3 |
217.3 |
17.0 |
13.7 |
7.0 |
6.0 |
15.8 |
18.7 |
32.7 |
88.0 |
91.3 |
231.3 |
243.7 |
15.7 |
12.7 |
7.3 |
8.7 |
50 |
16.0 |
34.3 |
97.7 |
130.3** |
253.7 |
252.3* |
17.7 |
13.3 |
9.3 |
10.7 |
158.1 |
24.0 |
41.3 |
123.0* |
152.3** |
248.3 |
297.3** |
15.0 |
11.7 |
10.3 |
8.0 |
500 |
32.3* |
93.0** |
162.3** |
267.3** |
324.3** |
414.0** |
8.7 |
12.0 |
4.0 |
7.0 |
1581 |
54.7** |
118.0** |
164.3** |
272.7** |
184.7 |
483.3** |
9.0 |
5.7 |
6.7 |
5.3 |
5000 |
53.7** |
81.0** |
154.7** |
231.7** |
161.3 |
490.5** |
6.0 |
7.0 |
6.0 |
6.7 |
Positive control |
583.3 |
351.7 |
504.3 |
984.7 |
612.0 |
1169.0 |
416.0 |
175.7 |
180.0 |
151.0 |
*p≤0.05
**statistically significant, p≤0.01
Table 2: Number of revertants per plate (mean of 3 plates (5 for negative control)) – Experiment 2
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
||||||
Conc. |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
-S-9 |
+S-9 |
0 (DMF) |
23.8 |
34.4 |
116.6 |
118.8 |
264.8 |
242.2 |
23.2 |
14.6 |
8.2 |
12.0 |
156.3 |
41.7** |
105.3** |
171.7** |
437.0** |
280.0 |
442.7** |
36.7 |
49.7** |
4.7 |
9.7 |
312.5 |
56.3** |
161.7** |
209.7** |
446.0** |
297.3 |
557.7** |
41.3* |
51.3** |
4.7 |
5.7 |
625 |
55.7** |
158.7** |
212.0** |
514.7** |
373.3** |
669.7** |
44.0** |
38.3** |
6.7 |
8.7 |
1250 |
63.0** |
210.3** |
205.7** |
612.7** |
361.3** |
662.7** |
34.7 |
45.3** |
4.3 |
9.7 |
2500 |
77.0** |
172.7** |
230.0** |
495.7** |
284.3 |
458.0** |
46.0** |
46.3** |
5.3 |
10.0 |
5000 |
66.7** |
159.7** |
190.0** |
423.0** |
203.3 |
278.7 |
27.3 |
45.7** |
3.0 |
7.7 |
Positive control |
923.7 |
384.7 |
665.7 |
1491.3 |
865.0 |
1520.0 |
580.0 |
302.7 |
104.0 |
173.7 |
*p≤0.05
**statistically significant, p≤0.01
Table 1. Results of a micronucleus test on Chinese hamster ovary cells
Treatment |
Concentration (µg/ml) |
Cytotoxicity (%)$ |
Mean MNBN cell frequency (%) |
Historical control range (%)# |
Statistical significance |
3+21-hour -S-9 |
Vehiclea |
- |
0.65 |
0.1-1.7 |
- |
20.00 |
9 |
1.20 |
p≤0.05 |
||
40.00 |
10 |
1.50 |
p≤0.01 |
||
70.00 |
19 |
1.75 |
p≤0.001 |
||
100.0 |
26 |
1.55 |
p≤0.01 |
||
*MMC, 0.80 |
ND |
3.80 |
p≤0.001 |
||
3+21 hour +S-9 |
Vehicle |
- |
0.60 |
0.3-1.8 |
- |
20.00 |
8 |
3.60 |
p≤0.001 |
||
40.00 |
19 |
3.45 |
p≤0.001 |
||
70.00 |
34 |
5.15 |
p≤0.001 |
||
100.0 |
43 |
5.00 |
p≤0.001 |
||
*CPA, 8.00 |
ND |
15.30 |
p≤0.001 |
||
24+0 hour -S-9 |
Vehicle |
- |
0.80 |
0.1-1.5 |
- |
10.00 |
6 |
0.85 |
NS |
||
20.00 |
6 |
1.25 |
NS |
||
40.00 |
12 |
0.65 |
NS |
||
70.00 |
12 |
0.95 |
NS |
||
*VIN, 0.005 |
ND |
0.85 |
NS |
a Vehicle control was 1% MC
* Positive control
# 95th percentile of the observed range
$ Based on replication index
NS Not significant
ND Not determined
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
The in vivo genotoxicity of dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 mg/kg bw/day of the test item on three consecutive days. Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues and micronuclei were analysed in bone marrow cells.
There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes. There was no increase in % tail intensity in the liver, glandular stomach, kidney or duodenum (Eurlings, 2020). As such, and as platinum was detected in the plasma of the test animals, the test item was considered to be non-genotoxic in vivo.
The data were considered relevant for di(tetraethylammonium)hexahydroxoplatinate via read-across (cfr. IUCLID section 13).
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Feb 2020 - 29 Jun 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:
19267COLPT.
- Expiration date of the lot/batch: 01 October 2021.
- Purity test date: CoA issued 17 December 2019.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.
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: 171 ± 8.7 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): 19 to 25°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.
IN-LIFE DATES:
From: Not specified.
To: 12 Mar 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. - 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.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Remarks:
- No treatment-related toxicity or mortality were seen in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 2000 mg/kg bw
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulphonate.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw, dissolved in physiological saline, administered twice. - Tissues and cell types examined:
- Cells were isolated from the liver, glandular stomach, duodenum and kidney.
- Details of tissue and slide preparation:
- Minced liver or kidney tissue was added to collagenase and dissolved in HBSS (saline). This suspension was shaken and centrifuged. The cell pellet was resuspended in HBSS and kept on ice prior to preparation of the slides.
Tissue from the glandular stomach and duodenum was stored on ice in "mincing buffer incomplete" (HBSS + EDTA). The surface epithelium of both the glandular stomach and duodenum was discarded as it contains a high proportion of apoptotic cells which distort the comet analysis. The cells, suspended in the buffer, were filtered though a 100 µm cell strainer and stored on ice prior to preparation of the slides.
Low melting point agarose was added to the cell suspensions and layered on a comet slide, which was then incubated for 13 - 39 minutes in the refrigerator.
Slides were kept overnight in the refrigerator, immersed in pre-chilled lysis solution. After rinsing, the slides were placed in freshly-prepared alkaline solution; electrophoresis was performed for 20 minutes (stomach and duodenum) or 30 minutes (liver and kidney). Following another rinse, the slides were immersed in absolute ethanol and allowed to dry, before staining with SYBR Gold fluorescent dye. - Evaluation criteria:
- A test item was considered positive if all of the following criteria were met:
a) at least one treatment group demonstrated a statistically significant increase in % tail intensity vs. control.
b) the increase was dose-related.
c) any of the results were outside the 95% confidence limits of the historical control data.
If none of the above criteria were met, 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. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Kidney: no statistically significant increase in % tail intensity.
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Liver: no statistically significant increase in % tail intensity.
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Glandular stomach: no statistically significant increase in % tail intensity.
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Duodenum: no statistically significant increase in % tail intensity.
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
Platinum was quantifiable in plasma samples from high-dose (2000 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 3-4 hours after the third dose. Therefore it was confirmed that the target tissues were exposed to the test item. No test item was detected in the animals dosed with vehicle.- Conclusions:
- When tested in the comet assay, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, did not induce an increase in DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 2000 mg/kg bw/day by gavage on three consecutive days. As such, this compound was considered to negative under the conditions of this assay.
- Executive summary:
The potential for dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received two doses of EMS (200 mg/kg bw/day). Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.
There was no increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item is not genotoxic to these tissues.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 11 Feb 2020 - 29 Jun 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- di(tetraethylammonium)hexahydroxoplatinate is considered to fall within the scope of the read-across "hexahydroxyplatinate(IV) compounds" approach (cfr. read-across justification report for mutagenicity (IUCLID section 13)).
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- 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
- 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: 171 ± 8.7 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): 19 to 25°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.
IN-LIFE DATES:
From: Not specified.
To: 12 Mar 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. - 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.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Remarks:
- No treatment-related toxicity or mortality were seen in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 2000 mg/kg bw
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulphonate.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw, dissolved in physiological saline, administered twice. - Tissues and cell types examined:
- Cells were isolated from the liver, glandular stomach, duodenum and kidney.
- Details of tissue and slide preparation:
- Minced liver or kidney tissue was added to collagenase and dissolved in HBSS (saline). This suspension was shaken and centrifuged. The cell pellet was resuspended in HBSS and kept on ice prior to preparation of the slides.
Tissue from the glandular stomach and duodenum was stored on ice in "mincing buffer incomplete" (HBSS + EDTA). The surface epithelium of both the glandular stomach and duodenum was discarded as it contains a high proportion of apoptotic cells which distort the comet analysis. The cells, suspended in the buffer, were filtered though a 100 µm cell strainer and stored on ice prior to preparation of the slides.
Low melting point agarose was added to the cell suspensions and layered on a comet slide, which was then incubated for 13 - 39 minutes in the refrigerator.
Slides were kept overnight in the refrigerator, immersed in pre-chilled lysis solution. After rinsing, the slides were placed in freshly-prepared alkaline solution; electrophoresis was performed for 20 minutes (stomach and duodenum) or 30 minutes (liver and kidney). Following another rinse, the slides were immersed in absolute ethanol and allowed to dry, before staining with SYBR Gold fluorescent dye. - Evaluation criteria:
- A test item was considered positive if all of the following criteria were met:
a) at least one treatment group demonstrated a statistically significant increase in % tail intensity vs. control.
b) the increase was dose-related.
c) any of the results were outside the 95% confidence limits of the historical control data.
If none of the above criteria were met, 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. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Kidney: no statistically significant increase in % tail intensity.
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Liver: no statistically significant increase in % tail intensity.
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Glandular stomach: no statistically significant increase in % tail intensity.
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Duodenum: no statistically significant increase in % tail intensity.
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Platinum was quantifiable in plasma samples from high-dose (2000 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 3-4 hours after the third dose. Therefore it was confirmed that the target tissues were exposed to the test item. No test item was detected in the animals dosed with vehicle.
- Conclusions:
- When tested in the comet assay, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, did not induce an increase in DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 2000 mg/kg bw/day by gavage on three consecutive days. As such, this compound was considered to negative under the conditions of this assay.
- Executive summary:
The potential for dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received two doses of EMS (200 mg/kg bw/day). Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.
There was no increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item is not genotoxic to these tissues.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Feb 2020 - 29 Jun 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline 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:
19267COLPT.
- Expiration date of the lot/batch: 01 October 2021.
- Purity test date: CoA issued 17 December 2019.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.
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: 171 ± 8.7 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): 19 to 25°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.
IN-LIFE DATES:
From: Not specified.
To: 12 Mar 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. - 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.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Remarks:
- No treatment-related toxicity or mortality were in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 2000 mg/kg bw
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide.
- Route of administration: Gavage.
- Doses / concentrations: A single dose of 19 mg/kg bw, dissolved in physiological saline. - Tissues and cell types examined:
- Bone marrow from the femur.
- Details of tissue and slide preparation:
- The femurs were flushed with foetal calf serum and the cell suspension centrifuged. The supernatant was removed and a drop of the remaining cell suspension was spread across a clean slide and fixed with methanol. The slides were automatically stained with Giemsa using the Wright Stain Procedure.
- Evaluation criteria:
- 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 8000 polychromatic erythrocytes per animal. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Platinum was quantifiable in plasma samples from high-dose (2000 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 3-4 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. - Conclusions:
- Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 2000 mg/kg bw/day by gavage on three consecutive days.
- Executive summary:
The in vivo clastogenicity and aneugenicity of dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 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, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, was concluded to be non-genotoxic under the conditions of this assay.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 11 Feb 2020 - 29 Jun 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- di(tetraethylammonium)hexahydroxoplatinate is considered to fall within the scope of the read-across "hexahydroxyplatinate(IV) compounds" approach (cfr. read-across justification report for mutagenicity (IUCLID section 13)).
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- 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
- 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: 171 ± 8.7 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): 19 to 25°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.
IN-LIFE DATES:
From: Not specified.
To: 12 Mar 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. - 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.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Remarks:
- No treatment-related toxicity or mortality were in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 2000 mg/kg bw
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide.
- Route of administration: Gavage.
- Doses / concentrations: A single dose of 19 mg/kg bw, dissolved in physiological saline. - Tissues and cell types examined:
- Bone marrow from the femur.
- Details of tissue and slide preparation:
- The femurs were flushed with foetal calf serum and the cell suspension centrifuged. The supernatant was removed and a drop of the remaining cell suspension was spread across a clean slide and fixed with methanol. The slides were automatically stained with Giemsa using the Wright Stain Procedure.
- Evaluation criteria:
- 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 8000 polychromatic erythrocytes per animal. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Platinum was quantifiable in plasma samples from high-dose (2000 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 3-4 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. - Conclusions:
- Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 2000 mg/kg bw/day by gavage on three consecutive days.
- Executive summary:
The in vivo clastogenicity and aneugenicity of dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 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, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, was concluded to be non-genotoxic under the conditions of this assay.
Referenceopen allclose all
Historical data Comet assay Negative control
|
Liver Males and Females |
Duodenum Males and Females |
Stomach Males and Females |
Kidney Males and Females |
Mean |
1.96 |
3.06 |
2.45 |
12.10 |
SD |
0.92 |
1.52 |
1.39 |
8.46 |
n |
85 |
45 |
60 |
30 |
Lower control limit (95% control limits) |
0.27 |
-0.86 |
-1.07 |
-1.35 |
Upper control limit (95% control limits) |
3.65 |
6.97 |
5.96 |
25.55 |
SD = Standard deviation
n = Number of observations
Kidney: Historical control data from experiments performed in Feb 2012 – July 2019
Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019
Historical data Comet assay Negative control
|
Liver Males and Females |
Duodenum Males and Females |
Stomach Males and Females |
Kidney Males and Females |
Mean |
1.96 |
3.06 |
2.45 |
12.10 |
SD |
0.92 |
1.52 |
1.39 |
8.46 |
n |
85 |
45 |
60 |
30 |
Lower control limit (95% control limits) |
0.27 |
-0.86 |
-1.07 |
-1.35 |
Upper control limit (95% control limits) |
3.65 |
6.97 |
5.96 |
25.55 |
SD = Standard deviation
n = Number of observations
Kidney: Historical control data from experiments performed in Feb 2012 – July 2019
Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Mode of Action Analysis / Human Relevance Framework
no data identified
Additional information
No studies conducted in humans were identified.
In an OECD Test Guideline 471 study, conducted according to GLP, di(tetraethylammonium)hexahydroxoplatinate was assessed for its ability to induce gene mutations in strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2 uvrA). Reproducible, large and dose-related increases in the number of revertant colonies, which were more than two-fold the control values, with TA98, TA100 and WP2 uvrA tester strains in the plate incorporation assays, both in the presence and absence of S9 metabolism were observed. It was concluded that di(tetraethylammonium)hexahydroxoplatinate was mutagenic in S. typhimurium and E.coli under the reported experimental conditions (Thompson, 2019).
No mammalian cell in vitro mutagenicity data are available for di(tetraethylammonium)hexahydroxoplatinate. Irrespective of the result of any cytogenicity test, the observation of mutagenic activity in bacterial cells (Thompson, 2019) necessitates further in vivo testing. Consequently, it was considered unnecessary to conduct further in vitro testing. Instead, see in vivo genotoxicity testing.
In a combined in vivo micronucleus test and Comet assay in rats, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, administered by gavage at doses of 500, 1000 or 2000 mg/kg bw/day for three days did not cause an increased incidence of micronucleated polychromatic erythrocytes. Treatment also gave no evidence of DNA damage in the liver, kidney, glandular stomach or duodenum when assessed by the Comet procedure. As such, the test item was considered to be non-genotoxic in vivo (Eurlings, 2020). These data are considered valid for di(tetraethylammonium)hexahydroxoplatinate via a read-across approach (cfr. IUCLID Section 13).
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 (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 (liability 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 carcinogenicity for other platinum compounds give no evidence of activity that would meet classification criteria (DECOS, 2008; SCOEL, 2011).
Following the generally positive in vitro results identified for the platinum compounds in various bacterial/mammalian cell mutagenicity assays (supported by some mammalian cell cytogenicity tests) and the unclear in vivo relevance of these in vitro findings, a combined in vivo micronucleus test and Comet assay in rats (with dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol) did not cause an increased incidence of micronucleated polychromatic erythrocytes and gave no evidence of DNA damage in the liver, kidney, glandular stomach or duodenum when assessed by the Comet procedure (Eurlings, 2020).
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, di(tetraethylammonium)hexahydroxoplatinate does not meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008).
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