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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).

The conclusion not to classify dihydrogen hexachloroplatinate as a germ cell mutagen should be revisited when the results of the planned in vivo studies (see testing proposal (IUCLID section 7.6.2) and read-across justification report for mutagenicity (IUCLID section 13)) are available.

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:
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

No in vivo genotoxicity data were identified.

 

The observation of mutagenic activity in bacterial cells (McGarry, 2013) and cytogenicity in mammalian cells (Lloyd, 2014) necessitates the consideration of further in vivo testing. Related platinum compounds have also demonstrated a general tendency to induce genotoxicity in vitro, though the available, if somewhat limited, dataset suggests that such findings might not be relevant in vivo and hence a similar lack of classification for mutagenicity is proposed for these compounds. Additional in vivo testing of certain related platinates has been proposed to further elucidate the in vivo relevance of the in vitro findings. The conclusion not to classify dihydrogen hexahydroxyplatinate as a germ cell mutagen should be revisited when the results of the planned in vivo studies (see testing proposals) are available.

Endpoint conclusion
Endpoint conclusion:
no study available

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).

 

No in vivo genotoxicity data were identified.

 

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).

 

Despite 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), the in vivo relevance of these in vitro findings remains unclear. Indeed, the available in vivo data returned mostly negative results. However, some of the identified studies might not be considered sufficiently robust (according to ECHA standards) to override the in vitro mutagenicity findings (e.g. a sex-linked recessive lethal test in Drosophila melanogaster (OECD TG 477, performed with dipotassium tetrachloroplatinate) and a liver unscheduled DNA synthesis assay (OECD TG 486, performed with tetraammineplatinum hydrogen carbonate)). Indeed, further in vivo testing of certain platinum compounds has been proposed to further elucidate the in vivo relevance of the in vitro findings (see testing proposal (IUCLID section 7.6.2) and read-across justification report for mutagenicity (IUCLID section 13)).

 

References

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

HCN (2008). Health Council of the Netherlands (DECOS). Platinum and platinum compounds. Health based recommended occupational exposure limit. https://www.gezondheidsraad.nl/sites/default/files/200812OSH_1.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 or 1B) under EU CLP criteria (EC 1272/2008). However, this conclusion should be revisited when the results of the planned in vivo studies are available.