[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: experimental study used as Weight of Evidence

Adequacy of study:

weight of evidence

Study period:

26 Feb 2020 - 30 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vivo genotoxicity data for similar Pt compounds are taken as weight-of-evidence to consider Pt concentrate P as non-genotoxic (see WoE argumentation in Section 13).

Cross-reference

Reason / purpose for cross-reference:

reference to same 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 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

Test material

Test material information

Constituent 1

Reference substance name:

Tetraammineplatinum dichloride

EC Number:

237-706-5

EC Name:

Tetraammineplatinum dichloride

Cas Number:

13933-32-9

Molecular formula:

Cl.1/2H12N4Pt

IUPAC Name:

Tetraammineplatinum dichloride

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.

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: 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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

-Vehicle(s)/solvent(s) used: corn oil (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 100, 104 and 106% of the nominal values for group 2, group 3 and group 4 (i.e. 250, 500 and 1000 mg/kg(bw)/d), respectively. Accuracy and homogeneity (coefficient of variation
≤ 10%) of the test item in vehicle was confirmed. "
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 mL/kg body weight
- Stability of test item in vehicle: Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10% of the initial mean sample concentration results. The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours (which exceeded the time between preparation and use of formulations).

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.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Maximum tolerable dose. In a preliminary dose-range finding study, 12 animals (group 1: 1 male and 1 female, group 2: 3 males and 3 females, Group 3: 1 male and 3 females) were dosed via oral gavage with 2000, 1000 and 1500 mg/kg body weight (groups 1, 2 and 3, respectively). Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg/day. These doses were therefore considered to be higher than the MTD. A dose of 1000 mg/kg/day was assumed to be the MTD.

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.

Examinations

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:

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.

The incidence of micronuclei was assessed in at least 4000 polychromatic erythrocytes per animal.


A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable for addition to the
laboratory historical control database
b) The concurrent positive controls should induce responses that are compatible with those
generated in the historical positive control database and produce a statistically significant
increase compared with the concurrent negative control. The positive control data was
analyzed by the Welch t test (inhomogeneous variances, one-sided, p < 0.05).
c) The appropriate number of doses and cells has been analysed.
d) The criteria for the selection of the highest dose are consistent with those described in the
OECD 474 guideline

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical
analysis of the micronucleus test data.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Confirmation of exposure:
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.

Characterisation data indicating whether micronuclei contain whole or fragmented chromosomes have not been generated since the in vivo MN assay was clearly negative. Moreover, this characterisation is no requirement in the OECD474 guidance.

Any other information on results incl. tables

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	0	2,0	± 1,6	1,24	± 0,14
			2	1
			3	3
			4	4
			5	2
2	test item	25	6	3	2,2	± 0,8	1,17	± 0,23
			7	2
			8	3
			9	1
			10	2
3	test item	50	11	4	2,6	± 1,9	1,16	± 0,19
			12	0
			13	2
			14	5
			15	2
4	test item	100	16	2	2,6	± 0,9	1,06	± 0,28
			17	2
			18	3
			19	4
			20	2
6	Cyclophosphamide	19	26	38	66,8	± 36,1 (4)	0,53	± 0,30
			27	31
			28	74
			29	69
			30	122
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 (Welch t test, P < 0.001).

 

Dose-response relationship & statistics

Micronucleus test (Evaluation of  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 Welsh 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 observations	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

Applicant's summary and conclusion

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.