Registration Dossier
Registration Dossier
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EC number: 240-973-0 | CAS number: 16919-58-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ammonium hexachloroplatinate was mutagenic in a bacterial reverse mutation (Ames) assay using four Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) when tested in the presence and absence of S9 (Bunger et al., 1996).
However, in a previously conducted Ames assay, ammonium hexachloroplatinate displayed no evidence of mutagenicity in S. typhimurium strains TA100, TA1535, TA1537 or TA1538 when tested at up to cytotoxic levels in the presence (TA100 and TA1537 only) or absence (all four strains) of S9. An inconsistent, weak positive result was seen in S. typhimurium TA98 in the presence, but not in the absence, of S9 (Bootman and May, 1980).
In a well-conducted in vitro mammalian gene mutation test using CHO cells, dipotassium hexachloroplatinate was weakly mutagenic when tested up to cytotoxic concentrations, in the absence of metabolic activation (Taylor et al., 1979).
Dipotassium hexachloroplatinate did not induce micronuclei in the cytokinesis-block micronucleus test with human lymphocytes, when tested at up to cytotoxic concentrations (Gebel et al., 1997).
However, according to a brief abstract, diammonium hexachloroplatinate was said to have shown a clear mutagenic effect, as indicated by the induction of a statistically higher number of micronuclei (MN) in two male donors than in controls in the dose-ranges of 75 -125 μM Pt. FISH analysis did not show a significant increase of MN-C (as a percentage), suggesting that the metal acts with both clastogenic and aneuploidogenic mechanisms (Migliore et al., 1999).
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
The in vivo genotoxicity of diammonium hexachloroplatinate, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes and to cause DNA damage, was assessed in a combined study following OECD 474 and 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The highest-tested dose was limited by clinical signs of toxicity, including mortalities, observed in a dose range finding study. Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues and micronuclei were analysed in bone marrow cells.
There was no increase in the number of micronucleated polychromatic erythrocytes in any treatment group. There was no increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item was not genotoxic to these tissues. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.
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:
- 01 May 2020 - 09 Jul 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Version / remarks:
- 29 July 2016.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian comet assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch number of test material: AI2707.
- Expiration date of the lot/batch: 25 August 2020 (from CoA).
- Purity: 99%.
- Purity test date: CoA issued 22 January 2020.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.
- Final preparation of a solid: Test item was suspended in corn oil.
FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspension. - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 150 ± 7.8 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.
IN-LIFE DATES:
From: Approx. 20 Mar 2020 (6 weeks before experimental start date).
To: 10 Jun 2020. - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands.
- Justification for choice of solvent/vehicle: corn oil is a widely used standard vehicle for in vivo animal experiments.
- Concentration of test material in vehicle: analytical verification confirmed that the measured test item concentrations in vehicle were 109%, 103% and 108% of the nominal values for group 2, group 3 and group 4 (i.e. 37.5, 75 and 105 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation ≤ 10%) of the test item in vehicle was confirmed.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Stability of test item in vehicle: stability of test item suspended in vehicle demonstrated for 4 hours at room temperature under normal laboratory conditions(sufficient for the dosing of all test animals), after which unused test item formulations were discarded. - Duration of treatment / exposure:
- Three consecutive days.
- Frequency of treatment:
- Daily.
- Post exposure period:
- Tissue samples taken 3 - 4 hours after administration of final dose.
- Dose / conc.:
- 37.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 75 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Remarks:
- Test item-related mortality was observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 200 mg/kg bw, and one animal of each sex received 300 mg/kg bw/day. Clinical signs of toxicity (ataxia, lethargy, hunched posture, rough coat and diarrhoea) were observed at 150 mg/kg bw/day, which was determined to be the maximum tolerated dose.
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulphonate.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw, dissolved in physiological saline, administered twice. - Tissues and cell types examined:
- Cells were isolated from the liver, glandular stomach, duodenum and kidney.
- Details of tissue and slide preparation:
- Minced liver or kidney tissue was added to collagenase and dissolved in HBSS (saline). This suspension was shaken and centrifuged. The cell pellet was resuspended in HBSS and kept on ice prior to preparation of the slides.
Tissue from the glandular stomach and duodenum was stored on ice in "mincing buffer incomplete" (HBSS + EDTA). The surface epithelium of both the glandular stomach and duodenum was discarded as it contains a high proportion of apoptotic cells which distort the comet analysis. The cells, suspended in the buffer, were filtered though a 100 µm cell strainer and stored on ice prior to preparation of the slides.
Low melting point agarose was added to the cell suspensions and layered on a pre-coated comet slide (Trevigen), which was then incubated for 10 - 21 minutes in the refrigerator. Three slides per tissue were prepared.
Slides were kept overnight in the refrigerator, immersed in pre-chilled lysis solution. After rinsing, the slides were placed in freshly-prepared alkaline solution; electrophoresis was performed for 20 minutes (stomach and duodenum) or 30 minutes (liver and kidney). Following another rinse, the slides were immersed in absolute ethanol and allowed to dry, before staining with SYBR Gold fluorescent dye. - Evaluation criteria:
- 150 comets were examined per sample using an IV image analysis system. Only horizontal comets, oriented with the head on the left and the tail on the right, were scored. Cells that showed overlap or were not sharp were not scored.
A test item was considered positive if all of the following criteria were met:
a) at least one treatment group demonstrated a statistically significant increase in % tail intensity vs. control.
b) the increase was dose-related.
c) any of the results were outside the 95% confidence limits of the historical control data.
If none of the above criteria were met, and direct or indirect evidence supportive of exposure of, or toxicity to, the target tissues was demonstrated, the test item was considered negative. If the data precluded making a conclusion of clearly positive or negative, the result was concluded as equivocal. - Statistics:
- ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical
analysis of the comet assay data .
A test item is considered positive in the comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p <
0.05) increase in percentage Tail Intensity is detected compared with the concurrent
negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in percentage Tail Intensity is detected compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Kidney: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Liver: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Glandular stomach: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Duodenum: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- One high-dose animal died after the first dose, and was replaced by an additional animal. Clinical signs of toxicity were observed in the high-dose group: hunched posture (4/5 animals), lethargy (5/5 animals) and diarrhoea (1/5 animals).
Platinum was quantifiable in plasma samples from high-dose (150 mg/kg bw/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3 hours after the third dose. Therefore it was confirmed that there was systemic exposure to the test item. No test item was detected in the animals dosed with vehicle. - Conclusions:
- When tested in the comet assay, diammonium hexachloroplatinate did not induce DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 150 mg/kg bw/day by gavage on three consecutive days. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.
- Executive summary:
The potential for diammonium hexachloroplatinate to cause DNA damage was evaluated in a study following OECD 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received two doses of EMS (200 mg/kg bw/day). Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.
There was no statistically significant increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item was not genotoxic to these tissues.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 May 2020 - 09 Jul 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch number of test material: AI2707.
- Expiration date of the lot/batch: 25 August 2020 (from CoA).
- Purity: 99%.
- Purity test date: CoA issued 22 January 2020.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.
- Final preparation of a solid: Test item was suspended in corn oil.
FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspension. - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 150 ± 7.8 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.
IN-LIFE DATES:
From: Approx. 20 Mar 2020 (6 weeks before experimental start date).
To: 10 Jun 2020. - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands.
- Justification for choice of solvent/vehicle: corn oil is a widely used standard vehicle for in vivo animal experiments.
- Concentration of test material in vehicle: analytical verification confirmed that the measured test item concentrations in vehicle were 109%, 103% and 108% of the nominal values for group 2, group 3 and group 4 (i.e. 37.5, 75 and 105 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation ≤ 10%) of the test item in vehicle was confirmed.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Stability of test item in vehicle: stability of test item suspended in vehicle demonstrated for 4 hours at room temperature under normal laboratory conditions(sufficient for the dosing of all test animals), after which unused test item formulations were discarded. - Duration of treatment / exposure:
- Three consecutive days.
- Frequency of treatment:
- Daily.
- Post exposure period:
- Tissue samples taken 3 - 4 hours after administration of final dose.
- Dose / conc.:
- 37.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 75 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Remarks:
- Test item-related mortality was observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 200 mg/kg bw, and one animal of each sex received 300 mg/kg bw/day. Clinical signs of toxicity (ataxia, lethargy, hunched posture, rough coat and diarrhoea) were observed at 150 mg/kg bw/day, which was determined to be the maximum tolerated dose.
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide.
- Route of administration: Gavage.
- Doses / concentrations: A single dose of 19 mg/kg bw, dissolved in physiological saline. - Tissues and cell types examined:
- Bone marrow from the femur.
- Details of tissue and slide preparation:
- The femurs were flushed with foetal calf serum and the cell suspension centrifuged. The supernatant was removed and a drop of the remaining cell suspension was spread across a clean slide and fixed with methanol. The slides were automatically stained with Giemsa using the Wright Stain Procedure.
The number of micronucleated polychromatic erythrocytes was initially counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).Slides were scored at a magnification of 1000x.
The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. - Evaluation criteria:
- The test item was considered positive if all of the following criteria were met:
a) at least one treatment group showed a statistically significant increase in frequency of micronucleated polychromatic erythrocytes.
b) the increase was dose related.
c) the results were outside the 95% confidence limits of the historical control data.
If none of the above criteria were met, and bone marrow exposure to the test item occurred, the substance was considered negative.
The incidence of micronuclei was assessed in 4000 polychromatic erythrocytes per animal. - Statistics:
- ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical
analysis of the data.
A test item is considered positive in the micronucleus test if all of the following criteria are
met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes
compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in the frequency of micronucleated polychromatic erythrocytes compared with
the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- One high-dose animal died after the first dose, and was replaced by an additional animal. Clinical signs of toxicity were observed in the high-dose group: hunched posture (4/5 animals), lethargy (5/5 animals) and diarrhoea (1/5 animals).
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Platinum was quantifiable in plasma samples from high-dose (150 mg/kg bw/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3 hours after the third dose. Therefore it was confirmed that the bone marrow was exposed to the test item. No test item was detected in the animals dosed with vehicle.
No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed.
Treated animals showed no decrease in the PCE:NCE ratio, indicating a lack of toxicity to the bone marrow. - Conclusions:
- Diammmonium hexachloroplatinate did not cause an increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of rats administered up to 150 mg/kg bw/day by gavage on three consecutive days. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.
- Executive summary:
The in vivo clastogenicity and aneugenicity of diammonium hexachloroplatinate, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes, was assessed in a study following OECD 474 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received a single dose of cyclophosphamide. Bone marrow was harvested from the femurs and assessed for micronuclei.
There was no increase in the number of micronucleated polychromatic erythrocytes in any treatment group. On that basis, diammonium hexachloroplatinate was concluded to be non-genotoxic under the conditions of this assay.
Referenceopen allclose all
| Group mean % tail DNA for the different tissues analyses (mean and standard deviation) | ||
| liver | tail intensity (%) | SD |
| vehicle control | 4.54 | 0.63 |
| test item 37.5 mg/kg | 4.70 | 0.74 |
| test item 75 mg/kg | 4.28 | 0.79 |
| test item 150 mg/kg | 3.76 | 0.23 |
| EMS 200 mg/kg | 82.11* | 6.52 |
| * significantly different (p<0.001) compared to corresponding vehicle control group | ||
| duodenum | tail intensity (%) | SD |
| vehicle control | 7.57 | 1.48 |
| test item 37.5 mg/kg | 6.64 | 1.30 |
| test item 75 mg/kg | 5.8 | 0.93 |
| test item 150 mg/kg | 6.15 | 0.78 |
| EMS 200 mg/kg | 46.84* | 4.76 |
| * significantly different (p<0.001) compared to corresponding vehicle control group | ||
| stomach | tail intensity (%) | SD |
| vehicle control | 6.32 | 1.57 |
| test item 37.5 mg/kg | 6.47 | 0.33 |
| test item 75 mg/kg | 4.22 | 0.83 |
| test item 150 mg/kg | 5.44 | 1.31 |
| EMS 200 mg/kg | 53.24* | 4.73 |
| * significantly different (p<0.001) compared to corresponding vehicle control group | ||
| kidney | tail intensity (%) | SD |
| vehicle control | 4.62 | 0.84 |
| test item 37.5 mg/kg | 4.96 | 1.84 |
| test item 75 mg/kg | 5.11 | 1.00 |
| test item 150 mg/kg | 5.16 | 0.59 |
| EMS 200 mg/kg | 86.05* | 4.45 |
| * significantly different (p<0.001) compared to corresponding vehicle control group | ||
| Historical data Comet assay Negative control | ||||
| Liver | Duodenum | Stomach | Kidney | |
| Tail Intensity (%) | Tail Intensity (%) | Tail Intensity (%) | Tail Intensity (%) | |
| Males and Females | Males and Females | Males and Females | Males and Females | |
| Mean | 2.4 | 4.3 | 3.5 | 9.1 |
| SD | 1.6 | 2.0 | 1.8 | 7.9 |
| n | 34 | 19 | 22 | 9 |
| Lower control limit (95% control limits) | -0.8 | 0.3 | 0.0 | -6.3 |
| Upper control limit (95% control limits) | 5.6 | 8.2 | 7.0 | 24.5 |
| SD = Standard deviation | ||||
| n = Number of observations | ||||
| Kidney: Historical control data from experiments performed in Feb 2012 – June 2020 | ||||
| Liver, Stomach, Duodenum: Historical control data from experiments performed in July 2017 – June 2020 | ||||
| Historical data Comet assay Positive control (200 mg/kg bw EMS orally dosed for two consecutive days) | ||||
| Liver | Duodenum | Stomach | Kidney | |
| Tail Intensity (%) | Tail Intensity (%) | Tail Intensity (%) | Tail Intensity (%) | |
| Males and Females | Males and Females | Males and Females | Males and Females | |
| Mean | 87.7 | 45.4 | 55.3 | 83.3 |
| SD | 6.7 | 12.1 | 11.6 | 11.8 |
| n | 33 | 19 | 22 | 9 |
| Lower control limit (95% control limits) | 74.5 | 21.7 | 32.6 | 60.2 |
| Upper control limit (95% control limits) | 100.9 | 69.1 | 78 | 106.4 |
| SD = Standard deviation | ||||
| n = Number of observations | ||||
| Kidney: Historical control data from experiments performed in Feb 2012 – June 2020 | ||||
| Liver, Stomach, Duodenum: Historical control data from experiments performed in July 2017 – June 2020 | ||||
| Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes | ||||||||
| group | treatment | Dose (mg/kg body weight) | animal number | Number of micronucleated polychromatic erythrocytes (number per animal) | Number of micronucleated polychromatic erythrocytes (mean +/- SD) (1,2) | ratio polychromatic/ normochromatic erythrocytes (mean +/- SD) (1,3) | ||
| 1 | vehicle control | 0 | 1 | 4 | 4.6 | ± 1.1 | 1.11 | ± 0.16 |
| 2 | 5 | |||||||
| 3 | 3 | |||||||
| 4 | 5 | |||||||
| 5 | 6 | |||||||
| 2 | test item | 37.5 | 6 | 0 | 1.6 | ± 1.7 | 1.22 | ± 0.06 |
| 7 | 0 | |||||||
| 8 | 2 | |||||||
| 9 | 2 | |||||||
| 10 | 4 | |||||||
| 3 | test item | 75 | 11 | 0 | 2.4 | ± 1.5 | 1.18 | ± 0.18 |
| 12 | 2 | |||||||
| 13 | 3 | |||||||
| 14 | 4 | |||||||
| 15 | 3 | |||||||
| 4 | test item | 150 | 16 | 2 | 3.2 | ± 2.2 | 1.0 | ± 0.08 |
| 17 | 0 | |||||||
| 18 | 4 | |||||||
| 19 | 5 | |||||||
| 21 | 5 | |||||||
| 6 | Cyclophosphamide | 19 | 29 | 8 | 10.8 | ± 1.8 (4) | 0.91 | ± 0.07 |
| 30 | 12 | |||||||
| 31 | 12 | |||||||
| 32 | 10 | |||||||
| 33 | 12 | |||||||
Legend
(1) Five animals per treatment group.
(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(3) The ratio was determined from at least the first 1000 erythrocytes counted.
(4) Significantly different from corresponding control group (Students t test, P < 0.001).
Dose-response relationship & statistics:
Test Item (comparison with the corresponding vehicle control group by using the Dunnett’s test): no significant differences
positive control: p-value (one sided) <0.001, significantly different from the corresponding vehicle control group by using the Student t-test
| Distribution historical control data from experiments performed between June 2017 and June 2020. | |||
| negative control data | positive control data | ||
| mean number of micronucleated cells per 4000 cells | 3.6 | 44.6 | |
| Standard deviation | 1.4 | 29.9 | |
| number of obsevations | 41 | 38 | |
| lower control limit (95% control limits) | 1 | -14 | |
| upper control limit (95% control limits) | 6 | 103 | |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Mode of Action Analysis / Human Relevance Framework
No data identified.
Additional information
Ammonium hexachloroplatinate was assessed for mutagenic activity in a bacterial reverse mutation
(Ames) assay, similar to OECD Test Guideline 471, using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 and tested at up to 500 μg/plate in the presence and absence of a metabolic activation system (S9) derived from phenobarbital- and beta-naphthoflavone-induced rat livers. (It is not clear whether the di- or mono-ammonium form was used; the recommended strain TA1535 was omitted.) Mutagenic effects were seen in all four strains both in the presence and absence of metabolic activation. Although no cytotoxicity data were provided for the test material, the minimum toxic dose for the platinum salts was apparently 100 μg/plate (Bunger et al., 1996). This study conforms to OECD guidelines, aside from the lack of inclusion of a fifth tester strain.
In a previously conducted study, the mutagenic activity of ammonium hexachloroplatinate in S. typhimurium was investigated in an Ames assay, similar to OECD Test Guideline 471, using pour-plate assays. The bacterial strains employed are capable of detecting both induced frameshift (TA1537, TA1538, and TA98) and base-pair substitution (TA1535 and TA100) mutations. Tests with strains TA98, TA100 and TA1537 were conducted at least in duplicate with the test material dissolved in deionised water at up to 1000 μg/plate (and in a subsequent test, due to inhibition of growth seen at the higher levels, at up to 100 μg/plate), both with and without S9. Two further tests (both in duplicate) were conducted in all five strains in the absence of S9, with the test material dissolved in isotonic saline solution at up to 100 μg/plate. No evidence of mutagenic activity was seen in any test with strains TA1535, TA1537, TA1538 or TA100, at up to cytotoxic levels, in the absence or presence of S9 (when used). A doubling of revertant colonies (weak positive) was seen in strain TA98 using the test material in deionised water at 40 μg/plate (test 1), and 4 and 20 μg/plate (test 2), in the presence of S9, but not in its absence. A further test with TA98 (test 3), again at up to 100 μg/plate (cytotoxic level), found no doubling in revertant colonies compared to spontaneous vehicle controls, with or without S9. Therefore, the study authors concluded that “no consistent evidence was obtained of mutagenic activity with strain TA98” (Bootman and May, 1980). This study does not entirely conform to current OECD guidelines which recommend including a bacterial strain which is susceptible to oxidative mutagenesis or cross-linking agents, for example S. typhimurium TA102, or Escherichia coli WP2 uvrA. Also, where the test material was dissolved in saline, no S9 was used, which is contrary to the OECD guidelines that recommend exposing bacteria to the test substance in the presence and absence of an appropriate metabolic activation system.
In a limited Ames spot test, diammonium hexachloroplatinate induced reverse mutations in S. typhimurium strain TA98 and E. coli strain WP2 hcr- try- when tested at up to 10 mM in the absence of metabolic activation (Kanematsu et al., 1980).
Dipotassium hexachloroplatinate was tested for genotoxicity in an in vitro mammalian gene mutation test using Chinese Hamster Ovary (CHO) cells deficient in HPRT (hypoxanthine-guanine phosphoribosyl transferase). Cells were tested only in the absence of a metabolic activation system. A 2-3-fold increase in the 8-AGR mutant frequency versus the spontaneous control was seen upon repeated subculture [prolonged exposure] with a non-cytotoxic concentration of 10 μM. This increase was apparent after 10 population doublings; the trend towards it was observed at 5 population doublings. Dipotassium hexachloroplatinate was determined to be weakly mutagenic to mammalian cells in the absence of metabolic activation under the conditions of the test (Taylor et al., 1979).
In a well-conducted study, similar to that described by OECD Test Guideline 487, the ability of dipotassium hexachloroplatinate to induce micronuclei in human peripheral mononuclear blood cells (lymphocytes) was assessed, in the absence of a metabolic activation system. The mean numbers of micronuclei in binucleate cells were 4, 5.5, 7.5 and 4 at test concentrations of 0, 5, 10 and 20 µM, respectively; there was no statistically significant change compared to the negative control. Severe cytotoxicity was observed at 40 µM. In conclusion, the test substance did not cause chromosome damage (micronuclei) in the cytokinesis-block micronucleus test with human lymphocytes (Gebel et al., 1997).
However, a brief abstract presents details of an in vitro micronucleus test using human lymphocytes from the whole blood of two young, non-smoking, males. Cells were blocked in cytokinesis by cytochalasin B. Micronuclei (MN) frequency was evaluated in binucleated lymphocytes and MN were analysed for the presence of a fluorescent signal by considering a labelled MN as centromere-positive MN (C+MN). Mytomycin (MMC, clastogen) and griseofulvin (GF, aneuploidogen) were used as positive standard mutagens. In addition, the fluorescence in situ hybridisation (FISH) technique with an alphoid centromere-specific DNA probe was applied in order to clarify the mechanism of action. Diammonium hexachloroplatinate was said to have shown a clear mutagenic effect, as indicated by the induction of a statistically higher number of MN in both donors than in controls in the dose-ranges of 75 -125 uM Pt. FISH analysis did not show a significant increase of MN-C (as a percentage), suggesting that the metal acts with both clastogenic and aneuploidogenic mechanisms (Migliore et al., 1999).
Dipotassium hexachloroplatinate did not cause DNA damage in a bacterial SOS chromotest, when tested at up to cytotoxic concentrations in the absence of metabolic activation (Gebel et al., 1997; Lantzsch and Gebel, 1997).
In a limited bacterial rec assay with ammonium hexachloroplatinate, the observed difference in inhibition of bacterial growth was described by the investigators as a strong positive rec effect, indicating possible damage DNA (Kanematsu et al., 1980).
In a combined rat micronucleus test and comet assay following OECD 474 and 489, there was no increase in the number of micronucleated polychromatic erythrocytes and no increase in % tail intensity in liver, kidney, glandular stomach or duodenum in rats given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days (Eurlings, 2020).
Several Expert Groups have assessed the toxicity profile of platinum, and various platinum compounds, including the assessment of CMR properties. All reviews have indicated that platinum compounds have been reported to be mutagenic in a range of in vitro studies (HCN, 2008; EMA, 2008; SCOEL, 2011; WHO, 1991). Cisplatin and related compounds are known DNA-reactive carcinogens and, as these compounds are better investigated due to their pharmaceutical properties, this has been confirmed in vivo. As cisplatin-type substances differ in chemical reactivity (liability of ligands, number of active sites etc.) it is reasonable to expect that not all forms of platinum are carcinogenic (HCN, 2008). Limited experimental data on carcinogenicity for other platinum compounds give no evidence of activity that would meet classification criteria (HCN, 2008; SCOEL, 2011).
Despite the generally positive in vitro results identified for the platinum compounds in various bacterial/mammalian cell mutagenicity assays (supported by some mammalian cell cytogenicity tests), the in vivo relevance of these in vitro findings remains unclear. Indeed, the new, high-quality in vivo data showed diammonium hexachloroplatinate itself to be conclusively non -genotoxic.
References
EMA (2008). European Medicines Agency. Guideline on the specification limits for residues of metal catalysts or metal reagents. Committee for Medicinal Products for Human Use (CHMP). EMEA/CHMP/SWP/4446/2000. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003586.pdf
HCN (2008). Health Council of the Netherlands (DECOS). Platinum and platinum compounds. Health based recommended occupational exposure limit. https://www.gezondheidsraad.nl/sites/default/files/200812OSH_1.pdf
SCOEL (2011). Recommendation from the Scientific Committee on Occupational Exposure Limits for platinum and platinum compounds. SCOEL/SUM/150. http://ec.europa.eu/social/BlobServlet?docId=7303&langId=en
WHO (1991). World Health Organization. Platinum. International Programme on Chemical Safety. Environmental Health Criteria 125. http://www.inchem.org/documents/ehc/ehc/ehc125.htm#SectionNumber:7.4
Justification for classification or non-classification
Based on the existing data set, diammonium hexachloroplatinate does not currently meet the criteria for classification as a germ cell mutagen (category 1A/1B or 2) under EU CLP criteria (EC 1272/2008).
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