Tetraammineplatinum dinitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genotoxicity of tetraammineplatinum dichloride, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes and to cause DNA damage, was assessed in a study following OECD 474 and 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 250, 500 or 1000 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.

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 or duodenum. 

There was a statistically significant and dose-related increase (p < 0.001) in DNA damage seen in the analysis of the kidney tissue. The tail intensity in animals dosed with 500 mg/kg bw/day was 14.56%, and in animals receiving 1000 mg/kg bw/day was 12.59%. However, these tail intensity values fell within the 95% confidence limits of the historical control data (upper limit 25.55%). Histopathological examination of the tissues did not reveal evidence of toxicity. As such, this finding was considered to be equivocal evidence of a genotoxic effect (Eurlings, 2020).

Link to relevant study records

Reference

Reference 1

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 - 30 Apr 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: 9005305492.
- Expiration date of the lot/batch: 07 January 2021.
- Purity test date: CoA issued 26 November 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (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: 138 ± 8.4 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: Not specified.
To: 09 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.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Maximum tolerable dose. Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg bw/day in a preliminary dose range finding study.

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, the test item was considered negative.

The incidence of micronuclei was assessed in at least 4000 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 (1000 mg/kg/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 animals were 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. A slight increase was seen in all treatment groups that was within the 95% limits of the historical control data.

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

Conclusions:

Tetraammineplatinum dichloride did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 1000 mg/kg bw/day by gavage on three consecutive days.

Executive summary:

The in vivo clastogenicity of tetraammineplatinum dichloride, 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 250, 500 or 1000 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 a slight but not statistically significant increase in micronucleated polychromatic erythrocytes in all treatment groups, but the incidences fell within the 95% limits of the historical control data. On that basis, tetraammineplatinum dichloride was concluded to be non-genotoxic under the conditions of this assay.

Reference 2

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 - 30 Apr 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:

yes

Remarks:

Only 3 or 4 animals were used for the isolation of the stomach in the control, low- and high-dose groups due to a technical error. All other values were within the historical controls and the results were clearly negative, so this did not impact the study

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:
9005305492.
- Expiration date of the lot/batch: 07 January 2021.
- Purity test date: CoA issued 26 November 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
In refrigerator (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: 138 ± 8.4 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: Not specified.
To: 09 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.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Maximum tolerable dose. Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg bw/day in a preliminary dose range finding study.

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 10 - 35 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, 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:

ambiguous

Remarks:

Kidney: Statistically significant and dose-related (p < 0.001 for the trend) increase in tail intensity, but the mean % tail intensity within the 95% limits of the historical control data. See table below

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Liver: no statistically significant increase in % tail intensity.

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

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

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:

Upper 95% confidence limit of historical control data: 25.55% tail intensity.
Platinum was quantifiable in plasma samples from high-dose (1000 mg/kg/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 animals were exposed to the test item. No test item was detected in the animals dosed with vehicle.

Comet results for kidney.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	3.52%	± 0.72%
250 mg/kg bw/day	7.56%	± 3.00%
500 mg/kg bw/day	14.56%	± 4.58%
1000 mg/kg bw/day	12.59%	± 6.09%
EMS (positive control)	79.86%	± 4.58%

Historical data Comet assay Negative control

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Stomach Tail Intensity (%) Males and Females	Kidney Tail Intensity (%) 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

Conclusions:

When tested in the comet assay, tetraammineplatinum dichloride did not induce an increase in DNA damage in the liver, glandular stomach or duodenum of rats administered up to 1000 mg/kg bw/day by gavage on three consecutive days. A statistically significant and dose-related (p < 0.001 for the trend) increase in DNA damage was seen in kidney cells, but the mean % tail intensity fell within the 95% limits of the historical control data. As such, this finding was considered to be equivocal evidence of a genotoxic effect.

Executive summary:

The potential for tetraammineplatinum dichloride 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 250, 500 or 1000 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, glandular stomach or duodenum, indicating that the test item is not genotoxic to these tissues.

There was a statistically significant and dose-related increase (p < 0.001) in DNA damage seen in the analysis of the kidney tissue. The tail intensity in animals dosed with 500 mg/kg bw/day was 14.56%, and in animals receiving 1000 mg/kg bw/day was 12.59%. However, these tail intensity values fell within the 95% confidence limits of the historical control data (upper limit 25.55%). As such, this finding was considered to be equivocal evidence of a genotoxic effect. No toxicity was observed in histopathological examination of the kidney tissues, indicating that this can be excluded as an indirect cause of the reported DNA damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

In a combined in vivo micronucleus test and Comet assay in rats, tetraammineplatinum dichloride administered by gavage at doses of 250, 500 or 1000 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, glandular stomach or duodenum when assessed by the Comet procedure. Analysis of the kidney tissue showed evidence of a statistically-significant and dose-related increase in % tail intensity. However, this increase fell within the historical control ranges and was therefore considered as equivocal evidence of a genotoxic effect (Eurlings, 2020).

 

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

 

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

 

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

 

Following the generally positive in vitro results identified for the tetraammineplatinum 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 tetraammineplatinum dichloride) did not cause an increased incidence of micronucleated polychromatic erythrocytes and gave no evidence of DNA damage in the liver, glandular stomach or duodenum when assessed by the Comet procedure. However, analysis of the kidney tissue showed evidence of a statistically-significant and dose-related increase in % tail intensity. Nevertheless, this increase fell within the historical control ranges and was therefore considered as equivocal evidence of a genotoxic effect (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, tetraammineplatinum nitrate does not currently meet the criteria for classification as a germ cell mutagen (category 1A/1B or 2).