[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation (Ames) test, conducted according to OECD Test Guideline 471 and to GLP, Pt concentrate P induced reverse mutations in strains of Salmonella typhimurium and Escherichia coli both in the absence and presence of a rat liver metabolic activation system (S9).

In a OECD TG 487 micronucleus study, to GLP, Pt concentrate P failed to induce biologically significant increases in the frequency of micronuclei.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 4133/00-K0
- Expiration date of the lot/batch: 07 June 2021
- Purity test date: Certificate of analysis dated 04 December 2020
- Form: liquid
- Appearance: Clear yellow liquid

Target gene:

Histidine for S. typhimurium strains; tryptophan for E.coli WP2 uvrA

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
The S9 batch is characterised with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene (Sigma), which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assay was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The mutation experiment was a direct plate assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Vehicle / solvent:

Sterile distilled water

Negative solvent / vehicle controls:

yes

Positive control substance:

sodium azide

Remarks:

5 µg/plate for TA1535 without S9

Positive control substance:

other:

Remarks:

2.5 µg/plate for TA1537 without S9

Positive control substance:

2-nitrofluorene

Remarks:

10 µg/plate for TA1537 and TA98 without S9

Positive control substance:

methylmethanesulfonate

Remarks:

650 µg/plate for TA100 without S9

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

10 µg/plate for WP2uvrA without S9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 µg/plate for TA1535 and TA1537, 1 µg/plate for TA98 and TA100, 15 µg/plate for WP2uvrA with S9

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
• The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and unless the total number of revertants in tester strain TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
A test item is considered positive (mutagenic) in the test if:
• The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three times the concurrent control.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation Experiment: Direct Plate Assay
The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and
5000 μg/plate.
Precipitate
Precipitation of the test item on the plates was not observed in any tester strain.
Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains at the top dose level of 5000 μg/plate in the absence and presence of S9-mix.
Mutagenicity
In tester strain TA100, both in the absence and presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 5.1- fold the concurrent control. The increases were outside the historical control data range and the increases were more than two-fold the concurrent control.
In tester strain TA98, both in the absence and presence of S9-mix, the test item induced
dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 28 and 19- fold the concurrent control, respectively. The increases were outside the historical control data range and more than three-fold the concurrent control.
In tester strain WP2uvrA, both in the absence and presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 2.4 and 2.3- fold the concurrent control, respectively. The increases were within the historical control data range but more than two-fold the concurrent control.

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Pt concentrate P induced substantial increases in the frequency of TA100, WP2uvrA and TA98 revertant colonies both with and without metabolic activation (S9-mix). Under the conditions of this test Pt concentrate P was considered to be mutagenic.

Executive summary:

In an OECD Test Guideline 471 study, conducted according to GLP, Pt concentrate P was assessed for its ability to induce gene mutations in strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2 uvrA).

The test item induced dose-related and biologically relevant increases in the number of revertant colonies compared to the solvent control in tester strain TA100, TA98 and WP₂uvrA.

No second experiment, including a pre-incubation step, was performed as the OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment.

 

It was concluded that Pt concentrate P was mutagenic in S. typhimurium and E.coli under the reported experimental conditions.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

Range-finder test with and without S9 (3+21-hour treatments): 156, 313, 625, 1250, 2500 and 5000 µg test item/mL

First Cytogenetic Assay
Without S9-mix: 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (3 hours exposure time, 27 hours harvest time)
With S9-mix: 100, 750, 1000, 1250, 1500, 2000, 2500 and 3000 µg/mL (3 hours exposure time, 2 7 hours harvest time)

Second Cytogenetic Assay
Without S9-mix: 100, 300, 500, 700, 900, 1100 and 1300 µg/mL (24 hours exposure time, 24 hours harvest time)

Vehicle / solvent:

Milli-Q water

Positive control substance:

cyclophosphamide

Remarks:

With Metabolic Activation; final concentration of 15 and 17.5 µg/mL for a 3 hour exposure period

Positive control substance:

colchicine

Remarks:

Without Metabolic Activation; final concentration of 0.1 µg/mL for a 3 hour exposure period and 0.05 µg/mL for a 24 hour exposure period

Positive control substance:

mitomycin C

Remarks:

Without Metabolic Activation; final concentration of 0.25 and 0.38 µg/mL for a 3 hour exposure period and 0.15 and 0.23 µg/mL for a 24 hour exposure period

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage 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 (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Statistics:

Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest concentration analyzed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The ability of the test item to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%.
The cytokinesis-block proliferation indices of cultures treated with various the test item concentrations or with the negative control items are presented in Table 1, Table 2, Table 4 and Table 6 . The scores for the number of binucleated cells with micronuclei are presented in Table 3, Table 5 and Table 7. Duplicate cultures are indicated by A and B.
The positive control chemicals produced a statistically significant increase in the number of binucleated cells with micronuclei. The number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
First cytogenetic assay
In the first cytogenetic assay, in the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.
In the absence of S9-mix, at the 3 hours exposure time, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest dose level. A dose related response was observed (Cochran Armitage trend test p = 0.0029). This increase was just above the 95% control limits of the distribution of the historical vehicle control database. Subsequent rescoring (quality control) of the same slides for the highest concentration by other slide scorers resulted in equivocal results. To elucidate these equivocal results, a repeat experiment was scheduled.
In the repeat experiment, clear negative results were obtained. The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix.
Second cytogenetic assay
In the second cytogenetic assay with a 24 hours continuous exposure time, the test item did not induce a dose dependent or statistically significant increase in the number of binucleated cells with micronuclei.

Table 1          
Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Pt concentrate P in the Dose-range Finding Test

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	232	161	107	1.75			0
156	315	118	67	1.50			33
313	268	162	75	1.62			18
625	315	145	40	1.45			40
1250	368	137	4	1.28			62
2500	456	43	1	1.09			88
5000	480	24	0	1.05			94

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	262	21	117	1.64			0
156	0	0	0	-			-
313	277	169	60	1.57			10
625	277	153	70	1.59			8
1250	324	147	29	1.41			36
2500	394	104	2	1.22			66
5000	442	75	0	1.15			77

 

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	254	149	97	1.69			0
156	309	135	56	1.49			28
313	316	138	48	1.47			32
625	332	162	20	1.39			43
1250	417	80	3	1.17			75
2500	453	58	0	1.11			83
5000	484	30	0	1.06			91

 

Table 2          
Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with Pt concentrate P in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.80	-	1.94	1.87		0
100	1.83	-	1.84	1.84		4
250	1.62	-	1.66	1.64		26
500	1.56	-	1.57	1.56		35
750	1.41	-	1.48	1.44		49
1000	1.35	-	1.37	1.36		58
1250	1.30	-	1.34	1.32		63
1500	1.19	-	1.21	1.20		77
2000	1.12	-	1.12	1.12		86
0.25 MMC-C	1.27	-	1.29	1.28		68
0.38 MMC-C	1.14	-	1.17	1.16		82
0.1 Colch	1.02	-	1.04	1.03		97
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.74	-	1.92	1.83		0
100	1.73	-	1.78	1.75		9
750	1.57	-	1.68	1.63		24
1000	1.70	-	1.71	1.70		15
1250	1.43	-	1.45	1.44		47
1500	1.33	-	1.47	1.40		52
2000	1.21	-	1.29	1.25		70
2500	1.15	-	1.22	1.18		78
3000	1.07	-	1.15	1.11		87
15 CP	1.16	-	1.18	1.17		79
17.5 CP	1.14	-	1.15	1.14		83

 

Table 3          
Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Pt concentrate P in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	2	1	3
100	4	0	2	2
500	35	1	0	1
1000	58	7	6	13^*2)
0.25 MMC-C	68	26	20	46^****
0.1 Colch	97	38	42	80^****

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	0	1	1
100	9	0	1	1
1250	47	0	2	2
1500	52	2	3	5^3)
15 CP	79	16	19	35^****

*)   Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

¹⁾   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾    Rescoring resulted in 14 and 4 micronucleated binucleated cells

³⁾    Rescoring resulted in 20 and 5 micronucleated binucleated cells

 

Table 4          
Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with Pt concentrate P in the Cytogenetic Assay 1A

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.52	-	1.65	1.59		0
100	1.50	-	1.51	1.51		14
500	1.37	-	1.39	1.38		36
650	1.25	-	1.31	1.28		52
800	1.21	-	1.22	1.22		63
950	1.15	-	1.17	1.16		73
1100	1.12	-	1.15	1.13		77
1350	1.14	-	1.14	1.14		77
1500	1.09	-	1.09	1.09		84
0.25 MMC-C	1.24	-	1.26	1.25		58
0.38 MMC-C	1.15	-	1.17	1.16		73
0.1 Colch	1.01	-	1.02	1.02		97
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.62	-	1.64	1.63		0
100	1.45	-	1.53	1.49		23
950	1.31	-	1.36	1.34		46
1100	1.28	-	1.28	1.28		55
1350	1.21	-	1.24	1.22		65
1500	1.16	-	1.21	1.19		71
1650	1.15	-	1.16	1.15		76
1800	1.14	-	1.16	1.15		77
1950	1.09	-	1.09	1.09		86
15 CP	1.05	-	1.06	1.05		92
17.5 CP	1.03	-	1.03	1.03		96

 

Table 5          
Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Pt concentrate P in Cytogenetic Assay 1A

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	0	1	1
100	14	0	0	0
500	36	3	3	6
650	52	1	1	2
0.25 MMC-C	58	12	13	25^****
0.1 Colch	97	39	38	77^****

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	0	6	6
100	9	0	2	2
1250	47	2	2	4
1500	52	0	2	2
15 CP	79	30	22	52^****

*)   Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

¹⁾   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

 

Table 6          
Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Pt concentrate P in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.82	-	1.84	1.83		0
100	1.68	-	1.74	1.71		14
300	1.49	-	1.65	1.57		32
500	1.45	-	1.60	1.53		37
700	1.39	-	1.46	1.43		49
900	1.32	-	1.38	1.35		58
1100	1.31	-	1.33	1.32		62
1300	1.28	-	1.30	1.29		65
0.15MMC-C	1.20	-	1.27	1.24		71
0.23MMC-C	1.14	-	1.15	1.14		83
0.05 Colch	1.00	-	1.00	1.00		100

 

Table 7          
Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Pt concentrate P in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	1	3	4
100	14	0	1	1
500	37	0	2	2
900	58	3	0	3
0.15 MMC-C	71	32	42	74^****
0.05 Colch	100	3^2)	2^2)	5^*

*)   Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

¹⁾    1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾   255 and 198 binucleated cells were scored for the presence of micronuclei, respectively.

 

 

 

Conclusions:

Negative. Pt concentrate P failed to induce biologically significant increases in the frequency of micronuclei in a OECD TG 487 micronucleus study.

Executive summary:

In an OECD Test Guideline 487 study, conducted according to GLP, Pt concentrate P was assessed for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.

The test item did not induce a statistically significant and biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the experiments.

In conclusion, Pt concentrate P is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genotoxicity of Diammonium hexachloroplatinate(IV), Dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2), Dipotassium tetrachloroplatinate and Tetraammineplatinum dichloride has been assessed in combined in vivo micronucleus (OECD 474) and Comet assays (OECD 489) according to GLP.

The result from these studies is used as weight-of-evidence to conclude that Pt concentrate P can be considered non-genotoxic (see weight-of-evidence argumentation in Section 13).

 

Pt concentrate P in mimicked gastric bodyfluid (HCl) reacts to Pt(IV) with one of the main compounds being hexachloroplatinate.

The in vivo genotoxicity of diammonium hexachloroplatinate, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes and to cause DNA damage, was assessed in a combined study following OECD 474 and 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The highest-tested dose was limited by clinical signs of toxicity, including mortalities, observed in a dose range finding study. Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues and micronuclei were analysed in bone marrow cells. 

There was no increase in the number of micronucleated polychromatic erythrocytes in any treatment group. There was no increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item was not genotoxic to these tissues. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.

Link to relevant study records

Reference

Reference 1

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).

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 (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.

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. 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.

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.

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.

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

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 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 - 20 May 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).

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.

Reference 3

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:

11 Feb 2020 - 29 Jun 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).

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:
19267COLPT.
- Expiration date of the lot/batch: 01 October 2021.
- Purity test date: CoA issued 17 December 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.

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: 171 ± 8.7 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): 19 to 25°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: 12 Mar 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.:

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

Dose / conc.:

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

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

No treatment-related toxicity or mortality were in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 2000 mg/kg bw

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, 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.

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.

As there were statistically significant differences between one or more of the test item groups
and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test
whether there is a significant trend in the induction.

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 (2000 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 3-4 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	2	1.4	± 0.9	1.14	± 0.10
			2	2
			3	1
			4	0
			5	2
2	test item	500	6	5	2.8	± 1.9	1.04	± 0.15
			7	2
			8	4
			9	3
			10	0
3	test item	1000	11	1	2.4	± 1.1	1.21	± 0.21
			12	3
			13	4
			14	2
			15	2
4	test item	2000	16	2	3.4	± 1.1 (4)	1.22	± 0.15
			17	3
			18	4
			19	3
			20	5
6	Cyclophosphamide	19	26	47	27.8	± 13.1 (5)	0.45	± 0.09
			27	17
			28	15
			29	34
			30	26
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 (Williams’ t test).
	(5) Significantly different from corresponding control group (Welch t test for inhomogeneous).
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)		ratio polychromatic/ normochromatic erythrocytes (mean +/- SD) (1,3)
1	vehicle control	0	1	4	4.8	± 2.6	1.21	± 0.13
			2	9
			3	2
			4	4
			5	5
2	test item	500	6	12	7.0	± 3.3	1.07	± 0.18
			7	7
			8	8
			9	4
			10	4
3	test item	1000	11	2	5.4	± 2.2	1.19	± 0.22
			12	8
			13	6
			14	5
			15	6
4	test item	2000	16	5	5.4	± 1.1	1.14	± 0.16
			17	4
			18	7
			19	5
			20	6
6	Cyclophosphamide	19	26	47	27.8	± 13.1 (4)	0.45	± 0.09
	(no additional scoring!)		27	17
			28	15
			29	34
			30	26
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) The ratio was determined from at least the first 2000 erythrocytes counted except for the positive control
	group were at least 1000 cells were counted..
	(4) Significantly different from corresponding control group (Welch t test for inhomogeneous).

 

Dose-response relationship & statistics
Micronucleus test (Evaluation of initial 4000 cells)
group 4 (2000 mg/kg(bw): p-value (one sided) <0.001 (comparison with the corresponding vehicle control group by using the Willliams' t-test), significantly different from the corresponding vehicle control group
all groups: p 0.026 (Analysis of a dose-response using a linear trend test), significant
positive control: p-value (one sided) <0.001, significantly different from the corresponding vehicle control group by using the Welch t-test
Micronucleus test (Evaluation of data including additional scoring)
Test Item: Comparison with the corresponding vehicle control group by using the Williams’ t 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 negative and positive 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

 

Conclusions:

Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 2000 mg/kg bw/day by gavage on three consecutive days.

Executive summary:

The in vivo clastogenicity and aneugenicity of dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 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, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, was concluded to be non-genotoxic under the conditions of this assay.

Reference 4

Endpoint:

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

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).

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.
- 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: 27.1, 54.1 or 108 mg/g (corresponding to nominal concentrations of 25, 50 and 100 mg/mL)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Stability of test item in vehicle: homogeneity of test item suspended in vehicle demonstrated for 4 hours (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.:

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 tables 1 and 5 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. See table 2 below

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. See table 3 below

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Duodenum: no statistically significant increase in % tail intensity. See table 4 below

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

See tables 5 (negative control) and 6 (positive control), below, for historical control data.

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.

Table 1: Comet results (% tail intensity) 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%

 

Table 2: Comet results (% tail intensity) for liver.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	2.23%	± 0.43%
250 mg/kg bw/day	1.80%	± 0.44%
500 mg/kg bw/day	1.73%	± 0.25%
1000 mg/kg bw/day	1.80%	± 0.52%
EMS (positive control)	81.47%	± 1.67%

 

Table 3: Comet results (% tail intensity) for glandular stomach.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	3.14%	± 0.85%
250 mg/kg bw/day	4.44%	± 1.39%
500 mg/kg bw/day	4.37%	± 0.90%
1000 mg/kg bw/day	4.15%	± 0.35%
EMS (positive control)	52.70%	± 7.21%

 

Table 4: Comet results (% tail intensity) for duodenum.

Dose	% Tail Intensity	S.D.
0 mg/kg bw/day (vehicle control)	2.18%	± 0.28%
250 mg/kg bw/day	2.63%	± 0.85%
500 mg/kg bw/day	2.86%	± 0.93%
1000 mg/kg bw/day	2.47%	± 0.51%
EMS (positive control)	34.29%	± 4.51%

 

Table 5: 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

 

Table 6: Historical data Comet assay Positive control (200 mg/kg bw EMS orally dosed for two consecutive days)

	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	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)	79.70	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, 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.

Reference 5

Endpoint:

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

Type of information:

other: experimental study used as Weight of Evidence

Adequacy of study:

weight of evidence

Study period:

26 Feb 2020 - 20 May 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).

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 6

Endpoint:

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

Type of information:

other: experimental study used as Weight of Evidence

Adequacy of study:

weight of evidence

Study period:

11 Feb 2020 - 29 Jun 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).

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:
19267COLPT.
- Expiration date of the lot/batch: 01 October 2021.
- Purity test date: CoA issued 17 December 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.

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: 171 ± 8.7 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): 19 to 25°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: 12 Mar 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 88%, 94% and 100% of the nominal values for group 2, group 3 and group 4 (i.e. 500, 1000 and 2000 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation
≤ 10%) of the test item in vehicle was confirmed."
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Stability of test item in vehicle: stability of test item suspended in vehicle demonstrated for 4 hours at room temperature under normal laboratory conditions(sufficient for the dosing of all test animals), after which unused test item formulations were discarded.

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Daily.

Post exposure period:

Tissue samples taken 3 - 4 hours after administration of final dose.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

No treatment-related toxicity or mortality were seen in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 2000 mg/kg bw

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 13 - 39 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, 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.

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: 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:

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 (2000 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 3-4 hours after the third dose. Therefore it was confirmed that the target tissues were exposed to the test item. No test item was detected in the animals dosed with vehicle.

 

Group mean % tail DNA for the different tissues analyses (mean and standard deviation)
liver	tail intensity (%)	SD
vehicle control	2.22	0.62
test item 500 mg/kg	2.79	1.01
test item 1000 mg/kg	2.46	0.61
test item 2000 mg/kg	2.31	0.48
EMS 200 mg/kg	75.7*	2.19
* significantly different (p<0.001) compared to corresponding vehicle control group
duodenum	tail intensity (%)	SD
vehicle control	5.38	1.92
test item 500 mg/kg	4.81	0.29
test item 1000 mg/kg	5.49	1.29
test item 2000 mg/kg	5.39	0.59
EMS 200 mg/kg	40.20*	7.95
* significantly different (p<0.001) compared to corresponding vehicle control group
stomach	tail intensity (%)	SD
vehicle control	3.83	0.52
test item 500 mg/kg	4.04	0.46
test item 1000 mg/kg	4.49	1.69
test item 2000 mg/kg	4.80	1.38
EMS 200 mg/kg	44.08*	4.35
* significantly different (p<0.001) compared to corresponding vehicle control group
kidney	tail intensity (%)	SD
vehicle control	2.93	0.85
test item 500 mg/kg	2.62	0.83
test item 1000 mg/kg	2.59	0.43
test item 2000 mg/kg	2.86	0.29
EMS 200 mg/kg	77.12*	4.38
* 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, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, did not induce an increase in DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 2000 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 dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 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, glandular stomach or duodenum, indicating that the test item is not genotoxic to these tissues.

Reference 7

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:

01 May 2020 - 09 Jul 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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: AI2707.
- Expiration date of the lot/batch: 25 August 2020 (from CoA).
- Purity: 99%.
- Purity test date: CoA issued 22 January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.
- Final preparation of a solid: Test item was suspended in corn oil.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspension.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 150 ± 7.8 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.

IN-LIFE DATES:
From: Approx. 20 Mar 2020 (6 weeks before experimental start date).
To: 10 Jun 2020.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands.

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Daily.

Post exposure period:

Tissue samples taken 3 - 4 hours after administration of final dose.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Test item-related mortality was observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 200 mg/kg bw, and one animal of each sex received 300 mg/kg bw/day. Clinical signs of toxicity (ataxia, lethargy, hunched posture, rough coat and diarrhoea) were observed at 150 mg/kg bw/day, which was determined to be the maximum tolerated dose.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide.
- Route of administration: Gavage.
- Doses / concentrations: A single dose of 19 mg/kg bw, dissolved in physiological saline.

Tissues and cell types examined:

Bone marrow from the femur.

Details of tissue and slide preparation:

The femurs were flushed with foetal calf serum and the cell suspension centrifuged. The supernatant was removed and a drop of the remaining cell suspension was spread across a clean slide and fixed with methanol. The slides were automatically stained with Giemsa using the Wright Stain Procedure.

The number of micronucleated polychromatic erythrocytes was initially counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).Slides were scored at a magnification of 1000x.

The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

The test item was considered positive if all of the following criteria were met:
a) at least one treatment group showed a statistically significant increase in frequency of micronucleated polychromatic erythrocytes.
b) the increase was dose related.
c) the results were outside the 95% confidence limits of the historical control data.

If none of the above criteria were met, and bone marrow exposure to the test item occurred, the substance was considered negative.

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

Statistics:

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

A test item is considered positive in the micronucleus test if all of the following criteria are
met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes
compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in the frequency of micronucleated polychromatic erythrocytes compared with
the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One high-dose animal died after the first dose, and was replaced by an additional animal. Clinical signs of toxicity were observed in the high-dose group: hunched posture (4/5 animals), lethargy (5/5 animals) and diarrhoea (1/5 animals).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Platinum was quantifiable in plasma samples from high-dose (150 mg/kg bw/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3 hours after the third dose. Therefore it was confirmed that the bone marrow was exposed to the test item. No test item was detected in the animals dosed with vehicle.

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

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

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes
group	treatment	Dose (mg/kg body weight)	animal number	Number of micronucleated polychromatic erythrocytes (number per animal)	Number of micronucleated polychromatic erythrocytes (mean +/- SD) (1,2)		ratio polychromatic/ normochromatic erythrocytes (mean +/- SD) (1,3)
1	vehicle control	0	1	4	4.6	± 1.1	1.11	± 0.16
			2	5
			3	3
			4	5
			5	6
2	test item	37.5	6	0	1.6	± 1.7	1.22	± 0.06
			7	0
			8	2
			9	2
			10	4
3	test item	75	11	0	2.4	± 1.5	1.18	± 0.18
			12	2
			13	3
			14	4
			15	3
4	test item	150	16	2	3.2	± 2.2	1.0	± 0.08
			17	0
			18	4
			19	5
			21	5
6	Cyclophosphamide	19	29	8	10.8	± 1.8 (4)	0.91	± 0.07
			30	12
			31	12
			32	10
			33	12

Legend

(1) Five animals per treatment group.
(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(3) The ratio was determined from at least the first 1000 erythrocytes counted.
(4) Significantly different from corresponding control group (Students t test, P < 0.001).

 

Dose-response relationship & statistics:
Test Item (comparison with the corresponding vehicle control group by using the Dunnett’s test): no significant differences
positive control: p-value (one sided) <0.001, significantly different from the corresponding vehicle control group by using the Student t-test

 

Distribution historical control data from experiments performed between June 2017 and June 2020.
		negative control data	positive control data
	mean number of micronucleated cells per 4000 cells	3.6	44.6
	Standard deviation	1.4	29.9
	number of obsevations	41	38
	lower control limit (95% control limits)	1	-14
	upper control limit (95% control limits)	6	103

 

Conclusions:

Diammmonium hexachloroplatinate did not cause an increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of rats administered up to 150 mg/kg bw/day by gavage on three consecutive days. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.

Executive summary:

The in vivo clastogenicity and aneugenicity of diammonium hexachloroplatinate, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes, was assessed in a study following OECD 474 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received a single dose of cyclophosphamide. Bone marrow was harvested from the femurs and assessed for micronuclei.

 

There was no increase in the number of micronucleated polychromatic erythrocytes in any treatment group. On that basis, diammonium hexachloroplatinate was concluded to be non-genotoxic under the conditions of this assay.

Reference 8

Endpoint:

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

Type of information:

other: experimental study used as Weight of Evidence

Adequacy of study:

weight of evidence

Study period:

01 May 2020 - 09 Jul 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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: AI2707.
- Expiration date of the lot/batch: 25 August 2020 (from CoA).
- Purity: 99%.
- Purity test date: CoA issued 22 January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.
- Final preparation of a solid: Test item was suspended in corn oil.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspension.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 150 ± 7.8 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.

IN-LIFE DATES:
From: Approx. 20 Mar 2020 (6 weeks before experimental start date).
To: 10 Jun 2020.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands.
- Justification for choice of solvent/vehicle: corn oil is a widely used standard vehicle for in vivo animal experiments.
- Concentration of test material in vehicle: analytical verification confirmed that the measured test item concentrations in vehicle were 109%, 103% and 108% of the nominal values for group 2, group 3 and group 4 (i.e. 37.5, 75 and 105 mg/kg(bw) respectively). Accuracy and homogeneity (coefficient of variation ≤ 10%) of the test item in vehicle was confirmed.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Stability of test item in vehicle: stability of test item suspended in vehicle demonstrated for 4 hours at room temperature under normal laboratory conditions(sufficient for the dosing of all test animals), after which unused test item formulations were discarded.

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Daily.

Post exposure period:

Tissue samples taken 3 - 4 hours after administration of final dose.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Test item-related mortality was observed in a preliminary dose range finding study in which three male and three female rats received three consecutive daily doses of 200 mg/kg bw, and one animal of each sex received 300 mg/kg bw/day. Clinical signs of toxicity (ataxia, lethargy, hunched posture, rough coat and diarrhoea) were observed at 150 mg/kg bw/day, which was determined to be the maximum tolerated dose.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulphonate.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw, dissolved in physiological saline, administered twice.

Tissues and cell types examined:

Cells were isolated from the liver, glandular stomach, duodenum and kidney.

Details of tissue and slide preparation:

Minced liver or kidney tissue was added to collagenase and dissolved in HBSS (saline). This suspension was shaken and centrifuged. The cell pellet was resuspended in HBSS and kept on ice prior to preparation of the slides.

Tissue from the glandular stomach and duodenum was stored on ice in "mincing buffer incomplete" (HBSS + EDTA). The surface epithelium of both the glandular stomach and duodenum was discarded as it contains a high proportion of apoptotic cells which distort the comet analysis. The cells, suspended in the buffer, were filtered though a 100 µm cell strainer and stored on ice prior to preparation of the slides.

Low melting point agarose was added to the cell suspensions and layered on a pre-coated comet slide (Trevigen), which was then incubated for 10 - 21 minutes in the refrigerator. Three slides per tissue were prepared.

Slides were kept overnight in the refrigerator, immersed in pre-chilled lysis solution. After rinsing, the slides were placed in freshly-prepared alkaline solution; electrophoresis was performed for 20 minutes (stomach and duodenum) or 30 minutes (liver and kidney). Following another rinse, the slides were immersed in absolute ethanol and allowed to dry, before staining with SYBR Gold fluorescent dye.

Evaluation criteria:

150 comets were examined per sample using an IV image analysis system. Only horizontal comets, oriented with the head on the left and the tail on the right, were scored. Cells that showed overlap or were not sharp were not scored.


A test item was considered positive if all of the following criteria were met:
a) at least one treatment group demonstrated a statistically significant increase in % tail intensity vs. control.
b) the increase was dose-related.
c) any of the results were outside the 95% confidence limits of the historical control data.

If none of the above criteria were met, and direct or indirect evidence supportive of exposure of, or toxicity to, the target tissues was demonstrated, the test item was considered negative. If the data precluded making a conclusion of clearly positive or negative, the result was concluded as equivocal.

Statistics:

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

A test item is considered positive in the comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p <
0.05) increase in percentage Tail Intensity is detected compared with the concurrent
negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in percentage Tail Intensity is detected compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Kidney: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Liver: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Duodenum: no statistically significant increase in % tail intensity. cfr table under section 'Any other information on results incl. tables'

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

One high-dose animal died after the first dose, and was replaced by an additional animal. Clinical signs of toxicity were observed in the high-dose group: hunched posture (4/5 animals), lethargy (5/5 animals) and diarrhoea (1/5 animals).

Platinum was quantifiable in plasma samples from high-dose (150 mg/kg bw/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3 hours after the third dose. Therefore it was confirmed that there was systemic exposure to the test item. No test item was detected in the animals dosed with vehicle.

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

Conclusions:

When tested in the comet assay, diammonium hexachloroplatinate did not induce DNA damage in the liver, kidney, glandular stomach or duodenum of rats administered up to 150 mg/kg bw/day by gavage on three consecutive days. As such, and as platinum was detected in the plasma of the test animals, diammonium hexachloroplatinate was concluded to be non-genotoxic in vivo.

Executive summary:

The potential for diammonium hexachloroplatinate to cause DNA damage was evaluated in a study following OECD 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 37.5, 75 or 150 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received two doses of EMS (200 mg/kg bw/day). Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.

 

There was no statistically significant increase in % tail intensity in the liver, kidney, glandular stomach or duodenum, indicating that the test item was not genotoxic to these tissues. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

no data identified

Additional information

The mixed in vitro genotoxicity results make it difficult to define the mutagenicity of Pt concentrate P. 

Several Expert Groups have assessed the toxicity profile of platinum, and various platinum compounds, including the assessment of CMR properties. All reviews have indicated that platinum compounds have been reported to be mutagenic in a range of in vitro studies (DECOS, 2008; EMA, 2008; SCOEL, 2011; WHO, 1991). However, despite the often positive in vitro results identified for the platinum compounds in various bacterial/mammalian cell mutagenicity assays, the in vivo relevance of these in vitro findings is uncertain. Indeed, in vivo data showed several Pt(II) and Pt(IV) compounds, e.g. diammonium hexachloroplatinate, to be conclusively non-genotoxic.

This information and the observation that Pt concentrate P in mimicked gastric bodyfluid (HCl) reacts to Pt(IV) with one of the main compounds being hexachloroplatinate supports the conclusion to consider Pt concentrate P as non-genotoxic (see weight-of-evidence argumentation in Section 13). 

 

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 and provided argumentation, Pt concentrate P does not meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008).