Hexachloroplatinic acid

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium hexachloroplatinate

EC Number:

240-973-0

EC Name:

Diammonium hexachloroplatinate

Cas Number:

16919-58-7

Molecular formula:

Cl6Pt.2H4N

IUPAC Name:

diammonium hexachloroplatinate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical description: Orange Powder
- Batch Number: YS1
- Storage and Handling: stored at room temperature in the dark

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.

Test animals

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.

Administration / exposure

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): dosing volume was 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.
-A separate study was performed to develop and to validate an analytical method for the
quantitative analysis of Diammonium hexachloroplatinate (CAS 16919-58-7) in vehicle (corn
oil) (Charles River Labs, Test Facility Study No. 20213554). The analytical method was validated for the following parameters:
• Specificity
• Calibration curve
• Accuracy and Repeatability
• Limit of quantification
• Stability of the analytical system and end solutions
• Stability of stock solutions
For Concentration and Homogeneity Analysis, storage conditions were at room temperature and the Acceptance Criteria for concentration (mean sample concentration results within or equal to
± 15% suspensions of theoretical concentration) and for homogeneity (relative standard deviation (RSD) of concentrations of +/- 10% for each group) were met. For Stability Analysis (4 h at room temperature), the Acceptance Criteria (Mean sample concentration results after storage that is within or equal to ±10% of the initial mean sample concentration results) were met too. The validated ICP-MS/MS method is thus considered suitable for the quantitative analysis of the
test item, based on platinum, in vehicle, in the test item target concentration range of
0.1 – 200 mg test item/g.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.