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Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): dihydrogen hexahydroxyplatinate
- Substance type: no data
- Physical state: yellow powder
- Analytical purity: 99-100%
- Impurities (identity and concentrations) (ppm): carbon (795); (ppm with respect to metal): silver (137), aluminium (20), calcium (48), chromium (15), copper (20), iron (128), iridium (26), potassium (15), magnesium (6), manganese (3), molybdenum (8), sodium (714), nickel (28), osmium (29), palladium (74), rhodium (32), ruthenium (11), silicon (54), tellurium (2), zirconium (3)
- Composition of test material, percentage of components: 64.83% platinum
- Isomers composition: no data
- Purity test date: 28 May 2013
- Lot/batch No.: CZ0185
- Expiration date of the lot/batch: May 2018
- Stability under test conditions: no data
- Storage condition of test material: 15-25°C, protected from light

Method

Target gene:
Histidine locus
Species / strainopen allclose all
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.
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.