Dihydrogen hexahydroxyplatinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a good-quality Ames assay, conducted according to GLP and OECD Test Guideline 471, dihydrogen hexahydroxyplatinate displayed evidence of mutagenicity when tested in five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537), in the presence and absence of a rat liver metabolic activation (S9) system (McGarry, 2013).

 

In an in vitro mammalian cell micronucleus test, conducted according to GLP and OECD Test Guideline 487, dihydrogen hexahydroxyplatinate induced micronuclei in cultured Chinese hamster ovary (CHO) cells following treatment with metabolic activation (S9). Weak evidence for inducing micronuclei was seen in the absence of S9 (Lloyd, 2014).

 

No in vitro mammalian cell gene mutation data are available for dihydrogen hexahydroxyplatinate. Further in vitro testing in considered unnecessary in the light of the available in vivo genotoxicity test data.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July to 2 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, performed to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Minor deviations did not affect the overall interpretation of study findings or compromise integrity of the study

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (main study (Experiments 1 and 2))

Additional strain / cell type characteristics:

other: histidine-requiring

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100 and TA 102 (range-finding study)

Additional strain / cell type characteristics:

other: histidine-requiring

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Range-finding study: 0, 5, 15.8, 50, 158.1, 500, 1581 or 5000 µg/plate

Main study:
Experiment 1: 0, 5, 15.8, 50, 158.1, 500, 1581 or 5000 µg/plate
Experiment 2: 0, 156.3, 312.5, 625, 1250, 2500 or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that dihydrogen hexahydroxyplatinate was insoluble in several commonly used solvents including water, acetone, anhydrous analytical grade dimethyl sulphoxide (DMSO), ethanol, tetrahydrofuran and dimethylformamide (DMF). The test article formed a homogenous suspension at approximately 50 mg/mL in water and at approximately 299 mg/mL in DMSO, DMF and acetone.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

50 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate

Positive control substance:

mitomycin C

Remarks:

TA102, without S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

benzo(a)pyrene

Remarks:

TA98, with S-9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate (TA100, TA1535, TA1537), 20 µg/plate (TA102)

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA102, TA1535 and TA1537, with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min (Experiment 2, TA1535 and TA1537 with S-9)
- Exposure duration: 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): ~3 days

NUMBER OF REPLICATIONS: single test plates (range-finding study); triplicate (main study (Experiments 1 and 2))

SELECTION AGENT (mutation assays): histidine-free medium

DETERMINATION OF CYTOTOXICITY
- Method: other: background lawns examined for signs of toxicity (e.g. marked reduction in revertants compared to controls)

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p≤0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example
consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Dunnett's test was used to assess the probability of the observed results arising by chance. Results were considered statistically significant when p≤0.01.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in Experiments 1 and 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in Experiments 1 and 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in Experiments 1 and 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in Experiments 1 and 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in Experiments 1 and 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment 1 at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

statistically significant increase in revertants in Experiments 1 and 2, but not clearly concentration-related or reproducible

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in Experiment 2, with pre-incubation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment 1 from 500 or 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

statistically significant increase in revertants in Experiment 2, but not clearly concentration-related or reproducible

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment 1 from 500 or 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment 2, at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

See tables 1 and 2 for revertant numbers/plate for experiments 1 and 2.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: noted in all strains treated with 5000 µg/plate, with and without S-9 (in Experiments 1 and 2)
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no evidence of cytotoxicity in tested strains (TA98, TA100 and TA102). Range-finding data were considered to be acceptable for cytotoxicity assessment only.

COMPARISON WITH HISTORICAL CONTROL DATA: results for vehicle controls were compared to historical control data from within the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was observed as a slight thinning of the background bacterial lawn, a marked reduction in revertant numbers, or a complete killing of the test bacteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Number of revertants per plate (mean of 3 plates (5 for negative control)) – Experiment 1

	TA98		TA100		TA102		TA1535		TA1537
Conc. (µg/plate)	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9
0 (DMF)	22.8	24.0	96.2	89.4	235.0	198.8	21.2	16.6	8.2	11.0
5	16.3	27.3	72.0	84.7	242.3	217.3	17.0	13.7	7.0	6.0
15.8	18.7	32.7	88.0	91.3	231.3	243.7	15.7	12.7	7.3	8.7
50	16.0	34.3	97.7	130.3**	253.7	252.3*	17.7	13.3	9.3	10.7
158.1	24.0	41.3	123.0*	152.3**	248.3	297.3**	15.0	11.7	10.3	8.0
500	32.3*	93.0**	162.3**	267.3**	324.3**	414.0**	8.7	12.0	4.0	7.0
1581	54.7**	118.0**	164.3**	272.7**	184.7	483.3**	9.0	5.7	6.7	5.3
5000	53.7**	81.0**	154.7**	231.7**	161.3	490.5**	6.0	7.0	6.0	6.7
Positive control	583.3	351.7	504.3	984.7	612.0	1169.0	416.0	175.7	180.0	151.0

*p≤0.05

**statistically significant, p≤0.01

 

Table 2: Number of revertants per plate (mean of 3 plates (5 for negative control)) – Experiment 2

	TA98		TA100		TA102		TA1535		TA1537
Conc. (µg/plate)	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9
0 (DMF)	23.8	34.4	116.6	118.8	264.8	242.2	23.2	14.6	8.2	12.0
156.3	41.7**	105.3**	171.7**	437.0**	280.0	442.7**	36.7	49.7**	4.7	9.7
312.5	56.3**	161.7**	209.7**	446.0**	297.3	557.7**	41.3*	51.3**	4.7	5.7
625	55.7**	158.7**	212.0**	514.7**	373.3**	669.7**	44.0**	38.3**	6.7	8.7
1250	63.0**	210.3**	205.7**	612.7**	361.3**	662.7**	34.7	45.3**	4.3	9.7
2500	77.0**	172.7**	230.0**	495.7**	284.3	458.0**	46.0**	46.3**	5.3	10.0
5000	66.7**	159.7**	190.0**	423.0**	203.3	278.7	27.3	45.7**	3.0	7.7
Positive control	923.7	384.7	665.7	1491.3	865.0	1520.0	580.0	302.7	104.0	173.7

*p≤0.05

**statistically significant, p≤0.01

Conclusions:

Interpretation of results (migrated information):
positive

In a good-quality Ames assay, conducted according to GLP and OECD Test Guideline 471, dihydrogen hexahydroxyplatinate displayed evidence of mutagenicity when tested in five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537), in the presence and absence of a rat liver metabolic activation (S9) system.

Executive summary:

Dihydrogen hexahydroxyplatinate was assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Guideline 471. Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were tested with and without the addition of a mammalian (rat liver) metabolic activation (S9) system in two separate experiments.

 

In the first experiment, agar containing the test substance at up to 5000 µg/plate was incubated with the bacterial strains for 3 days. The second experiment, also using concentrations of up to 5000 µg/plate, included an additional 20-minute pre-incubation step for cultures of TA1535 and TA1537 in the presence of S9.

 

There was significant evidence of mutagenicity in strains TA98 and TA100, with and without S9, and TA1535 and TA102 in the presence of S9 only. The lowest concentration producing a statistically significant increase in the number of revertant colonies was 50 µg/plate (for TA100, with S9). Cytotoxicity was observed at high concentrations in some of the test plates.Vehicle and positive controls performed as expected.

 

Under the conditions of this assay, dihydrogen hexahydroxyplatinate was mutagenic to S. typhimurium, with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genotoxicity of dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, 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 500, 1000 or 2000 mg/kg bw/day of the test item on three consecutive days. Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues and micronuclei were analysed in bone marrow cells.

 

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes. There was no increase in % tail intensity in the liver, glandular stomach, kidney or duodenum (Eurlings, 2020). As such, and as platinum was detected in the plasma of the test animals, the test item was considered to be non-genotoxic in vivo.

The data were considered relevant for dihydrogen hexahydroxyplatinate via read-across (cfr. IUCLID section 13).

 

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Feb 2020 - 29 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An EPMF member company received an official request from the Korean authorities to perform the in vivo mutagenicity assay with hexahydroxyplatinate,compound with 2-aminoethanol(1:2) (CAS 68133-90-4), ahead of the formal testing proposal approval by ECHA. The requested experimental information is in line with the assay submitted in the TP for this substance. The deadline given by the Korean authorities was however much tighter than the anticipated date of receiving the final decision on the TP. Therefore, the in vivo mutagenicity testing with this substance has been initiated in the EU prior to receiving the final decision on the TP.

In the attached document (translation from Korean and anonymised), a justification for initiating the in vivo muta testing upon official request from an non-EU authority (i.e. request outside EU-REACH), ahead of receiving the final decision on the TP. More information is available upon request.

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:
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 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.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Kidney: no statistically significant increase in % tail intensity.

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.

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.

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.

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.

Historical data Comet assay Negative control

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Stomach Tail Intensity (%) Males and Females	Kidney Tail Intensity (%) Males and Females
Mean	1.96	3.06	2.45	12.10
SD	0.92	1.52	1.39	8.46
n	85	45	60	30
Lower control limit (95% control limits)	0.27	-0.86	-1.07	-1.35
Upper control limit (95% control limits)	3.65	6.97	5.96	25.55

SD = Standard deviation

n = Number of observations

 

Kidney: Historical control data from experiments performed in Feb 2012 – July 2019

Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019

Conclusions:

When tested in the comet assay, 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 2

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

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:

For the group of hexahydroxyplatinate(IV) substances, it is proposed that, in aqueous solution and in biological media (e.g. gastric fluid), the cations (hydrogen or 2-aminoethanol) dissociate, leaving the core hexahydroxyplatinate(IV) complex as the common product and toxicologically-active species. It should be noted that this represents a chemical transformation and not a “biotransformation”; no metabolism of the hexahydroxyplatinate(IV) complex is anticipated to occur in vivo.
The 2-aminoethanol, acting as counter-ion of one group member, contributes to the effects observed for selected mammalian endpoints like irritation, repeated dose toxicity and reproductive toxicity. However, no contribution of 2-aminoethanol is expected for the mutagenicity endpoint. The mutagenicity test data should be considered equally applicable to each of the two group members.
More details on this read-across approach are provided in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

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

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 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.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Kidney: no statistically significant increase in % tail intensity.

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.

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.

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.

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.

Historical data Comet assay Negative control

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Stomach Tail Intensity (%) Males and Females	Kidney Tail Intensity (%) Males and Females
Mean	1.96	3.06	2.45	12.10
SD	0.92	1.52	1.39	8.46
n	85	45	60	30
Lower control limit (95% control limits)	0.27	-0.86	-1.07	-1.35
Upper control limit (95% control limits)	3.65	6.97	5.96	25.55

SD = Standard deviation

n = Number of observations

 

Kidney: Historical control data from experiments performed in Feb 2012 – July 2019

Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019

Conclusions:

When tested in the comet assay, 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 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

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:

An EPMF member company received an official request from the Korean authorities to perform the in vivo mutagenicity assay with hexahydroxyplatinate,compound with 2-aminoethanol(1:2) (CAS 68133-90-4), ahead of the formal testing proposal approval by ECHA. The requested experimental information is in line with the assay submitted in the TP for this substance. The deadline given by the Korean authorities was however much tighter than the anticipated date of receiving the final decision on the TP. Therefore, the in vivo mutagenicity testing with this substance has been initiated in the EU prior to receiving the final decision on the TP.

In the attached document (translation from Korean and anonymised), a justification for initiating the in vivo muta testing upon official request from an non-EU authority (i.e. request outside EU-REACH), ahead of receiving the final decision on the TP. More information is available upon request.

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.

Key result

Sex:

male

Genotoxicity:

negative

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.

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.

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.

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 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.

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 somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

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:

For the group of hexahydroxyplatinate(IV) substances, it is proposed that, in aqueous solution and in biological media (e.g. gastric fluid), the cations (hydrogen or 2-aminoethanol) dissociate, leaving the core hexahydroxyplatinate(IV) complex as the common product and toxicologically-active species. It should be noted that this represents a chemical transformation and not a “biotransformation”; no metabolism of the hexahydroxyplatinate(IV) complex is anticipated to occur in vivo.
The 2-aminoethanol, acting as counter-ion of one group member, contributes to the effects observed for selected mammalian endpoints like irritation, repeated dose toxicity and reproductive toxicity. However, no contribution of 2-aminoethanol is expected for the mutagenicity endpoint. The mutagenicity test data should be considered equally applicable to each of the two group members.
More details on this read-across approach are provided in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

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

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.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

No studies conducted in humans were identified.

 

Dihydrogen hexahydroxyplatinate was assessed for mutagenicity in a bacterial reverse mutation (Ames) assay performed to GLP, and according to OECD Guideline 471. Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were tested with and without the addition of a mammalian (rat liver) metabolic activation (S9) system in two separate experiments. In the first experiment, agar containing the test substance at up to 5000 µg/plate was incubated with the bacterial strains for 3 days. The second experiment, also using concentrations of up to 5000 µg/plate, included an additional 20-minute pre-incubation step for cultures of TA1535 and TA1537 in the presence of S9. There was significant evidence of mutagenicity in strains TA98 and TA100, with and without S9, and TA1535 and TA102 in the presence of S9 only. The lowest concentration producing a statistically significant increase in the number of revertant colonies was 50 µg/plate (for TA100, with S9). Cytotoxicity was observed at high concentrations in some of the test plates. Vehicle and positive controls performed as expected. Under the conditions of this assay, dihydrogen hexahydroxyplatinate was mutagenic to S. typhimurium, with and without metabolic activation (McGarry, 2013).

 

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 487 (in vitro mammalian cell micronucleus test), dihydrogen hexahydroxyplatinate was tested for its ability to induce chromosome damage in the form of micronuclei. Following two range-finding studies, Chinese hamster ovary (CHO) cells were treated with the test substance (in 1% methyl cellulose) for 3 hours (with a 21-hr recovery phase) in the presence and absence of rat liver (S9) metabolic activation, or for 24 hr in the absence of S9 alone. The maximum assessed concentrations, limited by precipitation of the test material, were 100 and 70 μg/ml for cells treated for 3 and 24 hr, respectively. For the 3-hr treatment in the presence of S-9, there was clear evidence of chromosome damage from the lowest tested concentration (20 µg/ml). In the absence of S9, results were ambiguous – statistically significantly higher incidences of micronuclei were observed relative to the vehicle control, but incidences were generally within the normal historical control range. The results from the 24-hour continuous treatment were not considered reliable; no significant genotoxicity was seen for the test substance or for the positive control. Nevertheless, results from the 3-hour treatment were considered sufficient to conclude that dihydrogen hexahydroxyplatinate is clastogenic under the conditions of this assay (Lloyd, 2014).

 

In a combined in vivo micronucleus test and Comet assay in rats, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, administered by gavage at doses of 500, 1000 or 2000 mg/kg bw/day for three days did not cause an increased incidence of micronucleated polychromatic erythrocytes. Treatment also gave no evidence of DNA damage in the liver, kidney, glandular stomach or duodenum when assessed by the Comet procedure. As such, the test item was considered to be non-genotoxic in vivo (Eurlings, 2020). These data are considered valid for dihydrogen hexahydroxyplatinate via a read-across approach (cfr. IUCLID Section 13).

 

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). Cisplatin and related compounds are known DNA-reactive carcinogens and, as these compounds are better investigated due to their pharmaceutical properties, this has been confirmed in vivo. As cisplatin-type substances differ in chemical reactivity (liability of ligands, number of active sites etc.) it is reasonable to expect that not all forms of platinum are carcinogenic (DECOS, 2008). Limited experimental data on carcinogenicity for other platinum compounds give no evidence of activity that would meet classification criteria (DECOS, 2008; SCOEL, 2011).

 

Following the generally positive in vitro results identified for the platinum compounds in various bacterial/mammalian cell mutagenicity assays (supported by some mammalian cell cytogenicity tests) and the unclear in vivo relevance of these in vitro findings, a combined in vivo micronucleus test and Comet assay in rats (with dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol) did not cause an increased incidence of micronucleated polychromatic erythrocytes and gave no evidence of DNA damage in the liver, kidney, glandular stomach or duodenum when assessed by the Comet procedure (Eurlings, 2020).

 

 

References

DECOS (2008). Dutch Expert Committee on Occupational Standards. Platinum and Platinum Compounds. Health-based recommended occupational exposure limit. Gezondheidsraad, 2008/12OSH. https://www.gezondheidsraad.nl/en/publications/gezonde-arbeidsomstandigheden/platinum-and-platinum-compounds-health-based-recommended

 

EMA (2008). European Medicines Agency. Guideline on the specification limits for residues of metal catalysts or metal reagents. Committee for Medicinal Products for Human Use (CHMP). EMEA/CHMP/SWP/4446/2000. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003586.pdf

 

SCOEL (2011). Recommendation from the Scientific Committee on Occupational Exposure Limits for platinum and platinum compounds. SCOEL/SUM/150. http://ec.europa.eu/social/BlobServlet?docId=7303&langId=en

 

WHO (1991). World Health Organization. Platinum. International Programme on Chemical Safety. Environmental Health Criteria 125.http://www.inchem.org/documents/ehc/ehc/ehc125.htm#SectionNumber:7.4

Justification for classification or non-classification

Based on the existing data set, dihydrogen hexahydroxyplatinate does not currently meet the criteria for classification as a germ cell mutagen (category 1A, 1B or 2) under EU CLP criteria (EC 1272/2008).