Dipotassium tetrachloroplatinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dipotassium tetrachloroplatinate displayed evidence of genotoxicity in several published in vitro gene mutation assays with bacterial and mammalian cells (Johnson et al., 1980; LeCointe et al., 1979; Suraikina et al., 1979; Uno and Morita, 1993) as well as in an in vitro micronucleus assay (Gebel et al., 1997) and two bacterial SOS chromotests (Gebel et al., 1997; Lantzsch and Gebel, 1997).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: chromosome damage (micronuclei)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although not a standard (guideline) study, it could be considered to be well documented and scientifically acceptable

Qualifier:

according to guideline

Guideline:

other: non standard method as described by Fenech M (1993) Mut Res 285, 35-44

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

Mammalian cytokinesis-block micronucleus assay similar to that descrobed by OECD TG487. Principal difference was that test was carried out only in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations of 0, 5, 15, 150 or 300 µM

Vehicle / solvent:

Distilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Briefly, blood from healthy donors (aged 25-35-years) was obtained, and the lymphocytes isolated, stained and counted. The lymphocytes were then cultured in medium at a concentration of 500,000/ml, and cell mitosis was stimulated. The test substance was disolved in distilled water and added 24 hr later to the culture in a volume of 20-30 µl. Seventy hours after cell mitosis was stimulated, the cells were harvested, fixed and prepared for microscopy. Micronuclei were scored in 1000 binucleate cells with two nuclei of equal size, and the nuclear division index (NDI) was calculated. Duplicate or triplicate experiments were carried out on different donors

Evaluation criteria:

Test substance was considered genotoxic if a statistically significant (p<0.05) increase in the mean number of micronuclei were observed.

Statistics:

Number of micronuclei analysed with the X2 test

Species / strain:

mammalian cell line, other: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity reported at 300 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean numbers of micronuclei in binucleate cells were 3.5, 3.5, 5.0 and 15.0 at concentrations of 0, 5, 15 and 150 µM, respectively. At 150 µM, this was statistically significant compared to the negative control. At 300 µM, cytotoxicity was seen.

Conclusions:

Interpretation of results (migrated information):
positive

Dipotassium tetrachloroplatinate induced micronuclei in the cytokinesis-block micronucleus test with human lymphocytes, when tested up to cytotoxic concentrations.

Executive summary:

In a non-guideline study, the ability of dipotassium tetrachloroplatinate 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 3.5, 3.5, 5.0 and 15.0 at test concentrations of 0, 5, 15 and 150 µM, respectively. At 150 µM, this was statistically significant compared to the negative control. At 300 µM, cytotoxicity was seen.

In conclusion, the test substance showed some ability to cause chromosome damage (micronuclei) in a cytokinesis-block micronucleus test with human lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Poorly reported study not conducted in accordance with modern protocol guidelines; results are not presented in a standard format. Nevertheless, the positive result from this study is useful in the overall weight of evidence.

Qualifier:

no guideline followed

Principles of method if other than guideline:

In a very limited Ames assay, tetraammine platinum dichloride was tested for its ability to induce mutations in a single strain of Salmonella typhimurium (TA100) in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Those involved in histidine biosynthesis.

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

A single concentration between 10 and 100 uM.

Vehicle / solvent:

No data

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

Apparently none employed.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

other: 0.5 revertants per nanomole were induced.

Additional information on results:

Results were presented graphically and the test compound induced between 1 and 10 revertants per nanomole (logarithmic scale, the indicator was closer to 1).

Conclusions:

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

Dipotassium tetrachloroplatinate was apparently mutagenic to a single strain of Salmonella typhimurium (TA100) in the absence of metabolic activation.

Executive summary:

In a very limited Ames assay, tetraammine platinum dichloride was tested for its ability to induce mutations in a single strain of Salmonella typhimurium (TA100) in the absence of metabolic activation. When tested at a concentration of between 10 and 100 uM, it induced between 1 and 10 revertants per nanomole, an apparently mutagenic effect (the positive control induced 0.5 revertants per nanomole).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genotoxicity of dipotassium tetrachloroplatinate, 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 25, 50 or 100 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. 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 or duodenum, indicating that the test item was not genotoxic to these tissues. A statistically significant increase in the mean tail intensity was observed in stomach cells of the low dose group (25 mg/kg bw/day). However, the increase was not dose related and no significant trend was observed. Therefore, the findings were considered not biologically relevant. As such, and as platinum was detected in the plasma of the test animals, dipotassium tetrachloroplatinate was concluded to be non-genotoxic in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 2020 - 20 May 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: 9005448302.
- Expiration date of the lot/batch: 16 September 2021.
- Purity test date: CoA issued 10 January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 - 8 °C).

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: 137 ± 8.6 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. 04 Feb 2020 (6 weeks before experimental start date).
To: 16 Apr 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 97% and 104% of the nominal values for group 2 and group 3 (i.e. 25 and 50 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation
≤ 10%) of the test item in vehicle was confirmed. Note that group 4 (100 mg/kg(bw) was dosed split-dose using the group3 dosing formulation with a 2-hour interval.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw except group 4 that was dosed split-dose and received 20 ml/kg. Also the vehicle control group was dosed split-dose and received 20 mL/kg.
- 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.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Test item-related clinical signs of toxicity were observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 100 mg/kg bw. This highest dose could only be administered split into two doses of 50 mg/kg bw administered 2 hours apart.

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 13 - 43 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.

cfr table under section 'Any other information on results incl. tables'

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.

cfr table under section 'Any other information on results incl. tables'

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:

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. cfr table under section 'Any other information on results incl. tables'

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach: statistically significant increase in % tail intensity in low-dose group only; not dose-related and no significant trend, so considered not biologically relevant cfr table under section 'Any other information on results incl. tables'

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. cfr table under section 'Any other information on results incl. tables'

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Platinum was quantifiable in plasma samples from high-dose (100 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.

A statistically significant increase in the mean tail intensity (%) was observed in stomach cells of the males in the low-dose (25 mg/kg bw/day) group (6.6%) which also exceeded the upper 95% control limit of the historical negative control database. However, the increase observed was not dose related and no significant trend was observed. Therefore, the findings were considered not biologically relevant.

Group mean % tail DNA for the different tissues analyses (mean and standard deviation)
liver	tail intensity (%)	SD
vehicle control	2.37	0.53
test item 25 mg/kg	1.77	0.41
test item 50 mg/kg	1.66	0.45
test item 100 mg/kg	2.17	0.83
EMS 200 mg/kg	90.04*	0.74
* significantly different (p<0.001) compared to corresponding vehicle control group
duodenum	tail intensity (%)	SD
vehicle control	3.57	1.09
test item 25 mg/kg	3.90	1.12
test item 50 mg/kg	3.38	1.01
test item 100 mg/kg	3.55	1.08
EMS 200 mg/kg	45.91*	7.00
* significantly different (p<0.001) compared to corresponding vehicle control group
stomach	tail intensity (%)	SD
vehicle control	4.20	0.73
test item 25 mg/kg	6.60*	1.23
test item 50 mg/kg	4.50	1.42
test item 100 mg/kg	4.80	1.68
EMS 200 mg/kg	59.68£	5.4
* significantly different (p<0.01) compared to corresponding vehicle control group using Dunnett's test. Analysis of dose-response using a linear trend test was not significant (p 0.455)
£ significantly different (p<0.001) compared to corresponding vehicle control group
kidney	tail intensity (%)	SD
vehicle control	2.83	0.30
test item 25 mg/kg	3.03	0.27
test item 50 mg/kg	2.88	0.45
test item 100 mg/kg	3.07	0.52
EMS 200 mg/kg	82.92*	4.71
* 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	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 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	89.53	41.17	55.16	84.92
SD	6.89	14.03	14.23	13.82
n	80	44	59	30
Lower control limit (95% control limits)	79.7	20.78	34.74	72.81
Upper control limit (95% control limits)	99.36	61.56	78.58	97.03
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

Conclusions:

When tested in the comet assay, dipotassium tetrachloroplatinate did not induce a biologically significant increase in DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 100 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 dipotassium tetrachloroplatinate 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 25, 50 or 100 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 or duodenum, indicating that the test item was not genotoxic to these tissues. A statistically significant increase in the mean tail intensity was observed in stomach cells of the low dose group (25 mg/kg bw/day) However, the increase was not dose related and no significant trend was observed. Therefore, the findings were considered not biologically relevant.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 2020 - 20 May 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: 9005448302.
- Expiration date of the lot/batch: 16 September 2021.
- Purity test date: CoA issued 10 January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 - 8 °C).

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: 137 ± 8.6 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. 04 Feb 2020 (6 weeks before experimental start date).
To: 16 Apr 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.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Test item-related clinical signs of toxicity were observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 100 mg/kg bw. This highest dose could only be administered split into two doses of 50 mg/kg bw administered 2 hours apart.

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%). Subsequently, a further 4000 cells were counted. 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 8000 polychromatic erythrocytes per animal.

cfr table under section 'Any other information on results incl. tables'

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.

cfr table under section 'Any other information on results incl. tables'

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

cfr table under section 'Any other information on results incl. tables'

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Platinum was quantifiable in plasma samples from high-dose (100 mg/kg bw/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3 hours after the third dose. Therefore it was confirmed that the bone marrow was exposed to the test item. No test item was detected in the animals dosed with vehicle.

No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed.

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

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic  Erythrocytes (scoring of 4000 initial cells for all groups including the positive control)
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	0	1.2	± 1.3	1.11	± 0.04
			2	2
			3	3
			4	0
			5	1
2	test item	25	6	1	2.0	± 1.0	1.12	± 0.04
			7	1
			8	3
			9	2
			10	3
3	test item	50	11	0	2.2	± 1.8	1.06	± 0.10
			12	2
			13	5
			14	2
			15	2
4	test item	100	16	0	1.8	± 2.6	1.08	± 0.08
			17	1
			18	4
			19	3
			20	1
6	Cyclophosphamide	19	26	25	26.0	± 8.2 (4)	0.54	± 0.25
			27	27
			28	38
			29	15
			30	25
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).
Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes (including scoring of 4000 additional cells for all groups except the positive control)
group	treatment	Dose (mg/kg body weight)	animal number	Number of micronucleated polychromatic erythrocytes (number per animal)	Number of micronucleated polychromatic erythrocytes (mean +/- SD) (1,2,3)		ratio polychromatic/ normochromatic erythrocytes (mean +/- SD) (1,4)
1	vehicle control	0	1	4	3.8	± 1.9	1.25	± 0.17
			2	3
			3	7
			4	2
			5	3
2	test item	25	6	8	5.2	± 1.8	1.22	± 0.06
			7	3
			8	5
			9	5
			10	5
3	test item	50	11	3	4.4	± 1.7	1.12	± 0.15
			12	3
			13	7
			14	4
			15	5
4	test item	100	16	2	3.6	± 1.8	1.23	± 0.16
			17	3
			18	6
			19	5
			20	2
6	Cyclophosphamide	19	26	25	26.0	± 8.2 (5)	0.54	± 0.25
	(no additional scoring!)		27	27
			28	38
			29	15
			30	25
legend	(1) Five animals per treatment group.
	(2) At least 8000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%, except for the
	positive control were at least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of
	5%.
	(3) Between brackets are the number of micronuclei in the vehicle control and test item groups converted to
	number of micronuclei per 4000 polychromatic erythrocytes
	(4) The ratio was determined from at least the first 2000 erythrocytes counted
	(5) Significantly different from corresponding control group (Students t test, P < 0.001).

 

Dose-response relationship & statistics
Micronucleus test (Evaluation of initial 4000 cells)
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
Micronucleus test (Evaluation of data including additional scoring)
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 November 2016 and November 2019.
		negative control data	positive control data
	mean number of micronucleated cells per 4000 cells	3.9	45.2
	Standard deviation	1.0	31.8
	number of obsevations	29	29
	lower control limit (95% control limits)	2*	-17
	upper control limit (95% control limits)	6	108
	legend: * Rounded value; unrounded value is 1.895

Conclusions:

Dipotassium tetrachloroplatinate did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 100 mg/kg bw/day by gavage on three consecutive days.

Executive summary:

The in vivo clastogenicity and aneugenicity of dipotassium tetrachloroplatinate, 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 25, 50 or 100 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, dipotassium tetrachloroplatinate was concluded to be non-genotoxic under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

Additional information from genetic toxicity in vivo:

In a limited Ames test, dipotassium tetrachloroplatinate was apparently mutagenic to a single strain of Salmonella typhimurium (TA100) when tested solely in the absence of metabolic activation (LeCointe et al., 1979).

 

Mutagenic activity was also observed in S. typhimurium strains TA98 and TA100 in the absence of metabolic activation (Suraikina et al., 1979).

 

A separate assay reported a weak mutagenic response in the same two strains, both in the presence and absence of S9 (Uno and Morita, 1993).

 

In a well-conducted in vitro mammalian gene mutation test using CHO cells, dipotassium tetrachloroplatinate was non-mutagenic when tested up to cytotoxic concentrations, in the absence of S9 (Taylor et al., 1979).

 

In a separate assay, ambiguous results were seen when dipotassium tetrachloroplatinate was tested at up to 65 μM in CHO cells in the absence of S9 (Johnson et al., 1980).

 

Dipotassium tetrachloroplatinate induced micronuclei in the cytokinesis-block micronucleus test with human lymphocytes, when tested up to cytotoxic concentrations (Gebel et al., 1997).

 

Evidence of DNA damage was observed when dipotassium tetrachloroplatinate was tested in bacterial SOS chromotests (Gebel et al., 1997; Lantzsch and Gebel, 1997).

 

No increase in sex-linked recessive lethal mutations was observed in the progeny of Drosophila melanogaster following oral administration of potassium chloro palatinate (II) at concentrations of 8 or 40 µg/kg bw/day (Bootman & Lodge, 1981).

 

Potassium chloroplatinite showed no evidence of clastogenicity in an in vivo assay for chromosome aberrations in bone marrow cells when administered to Chinese hamsters at up to 150 mg/kg bw/day for 5 consecutive days (Bootman & Whalley, 1981-1982).

 

Further, no evidence of clastogenicity was apparent in an in vivo micronucleus assay in mice after a single dose of up to 150 mg potassium chloroplatinite /kg bw (Bootman & Rees, 1981).

 

In a combined rodent 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 or duodenum in rats given gavage doses of 25, 50 or 100 mg/kg bw/day of the test item on three consecutive days. An increase in tail intensity in the stomach cells of the low-dose group was considered not biologically relevant (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 vitro (DECOS, 2008; EMA, 2008; SCOEL, 2011; WHO, 1991). Cisplatin and related compounds are known DNA-reactive carcinogens and, as these compounds are better investigated due to their pharmaceutical properties, this has been confirmed in vivo. As cisplatin-type substances differ in chemical reactivity (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 reproductive toxicity and carcinogenicity for other platinum compounds give no evidence of activity that would meet classification criteria (DECOS, 2008; SCOEL, 2011).

 

Despite the generally positive in vitro results identified for the platinum compounds in various bacterial/mammalian cell mutagenicity assays (supported by some mammalian cell cytogenicity tests), the in vivo relevance of these in vitro findings remains unclear. Indeed, the new, high-quality in vivo data showed dipotassium tetrachloroplatinate itself to be conclusively non -genotoxic.

 

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 selection of genetic toxicity endpoint
Well-conducted in vivo genotoxicity study, involving repeated dosing.

Justification for classification or non-classification

Based on the existing data set, dipotassium tetrachloroplatinate does not currently meet the criteria for classification as a germ cell mutagen (category 1A/1B or 2).