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Toxicological information

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:
11th April 2000 to 25th April 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dipotassium heptafluorotantalate
EC Number:
240-986-1
EC Name:
Dipotassium heptafluorotantalate
Cas Number:
16924-00-8
Molecular formula:
F7Ta.2K
IUPAC Name:
Tantalate(2-), heptafluoro-, potassium (1:2)
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Potassium fluorotantalate
- Substance type: White crystals
- Storage condition of test material: Room temperature

Method

Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
- Type and identity of media: Strains were kept in nutrient broth cultures and stored at -80ºC. Dimethyl sulphoxide was added to the cultures at 8% v/v as a cryopreservative.
- Properly maintained: yes
- Each batch was tested for cell membrane permeability, sensitivity to UV light and the pKM101 plasmid. Responses to a series of diagnostic mutagens were also tested.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Strains were kept in nutrient broth cultures and stored at -80ºC. Dimethyl sulphoxide was added to the cultures at 8% v/v as a cryopreservative.
- Properly maintained: yes
- Each batch was tested for cell membrane permeability, sensitivity to UV light and the pKM101 plasmid. Responses to a series of diagnostic mutagens were also tested.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5000, 1500, 500, 150, 50, 15 and 5 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% methyl cellulose
- Justification for choice of solvent/vehicle: Test material is insoluble in; water, dimethyl sulphoxide, ethanol, acetone and hexane. After grinding the test material with methyl cellulose an dosable suspension was formed.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% methyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: First test: in agar (plate incorporation). Second test: preincubation

First test
- 0.1 mL of a 10 hour bacterial culture and 0.5 mL S-9 mix or 0.5 mL 0.1 M sodium phosphate buffer (pH 7.4) were placed in glass bottles. 100 µL of the test solution was added, followed immediately by 2 mL of molten agar containing 0.05 mM histidine/biotin/tryptophan. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. All plates were incubated at 37 ºC for approximately 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted.

Second Test
- A second test was performed due to the negative response of the first test with a variation in procedure. A pre-incubation assay was used in which the bottles contained mixtures of bacteria, buffer or S9 mix and test solution were incubated at 37 ºC for 30 minutes with shaking before the addition of the agar overlay. Only 5 concentrations of the test material were used: 5000, 1500, 500, 150 and 50 µg/plate.

NUMBER OF REPLICATIONS: Each concentration was performed on 3 plates

EXAMINATIONS: After the total incubation period the plates were examined and colony numbers quantified for comparison.
Evaluation criteria:
- If the plates treated with the test material produce an increase in colony numbers of at least twice the concurrent vehicle controls, with evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in the test material.
- If exposure to the test material does not produce reproducible increase of at least 1.5 times the concurrent vehicle controls, in either mutation test, it is considered to show no mutagenic activity in the test material.
- If there is no clear positive response then the results will be subjected to statistical analysis.
Statistics:
The statistical analysis will be performed as described in Mahon et al (1989) and will normally be analysis of variance followed by a Dunnett's test.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation or thinning of background lawn was observed in either Test 1 or Test 2 at any test substance concentration in either the absence or presence of S9.
There were no significant increases in the number of revertant colonies compared to the vehicle control in either Test 1 or Test 2 at any test substance concentration in either the absence or presence of S9.

Full details of results for the test material (table 2) and positive controls (table 3) can be seen in the field "Any other information on results incl. tables".
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2. Revertant colony counts obtained per plate

Test Strain Concentration S9 mix Mean revertant colony counts SD
    (µg/plate)    
1 TA 1535 5000 - 11 4
  1500 - 13 6
  500 - 14 2
  150 - 15 5
  50 - 13 8
  15 - 13 2
  5 - 13 2
  Vehicle - 18 1
  5000 + 11 3
  1500 + 12 2
  500 + 17 5
  150 + 15 4
  50 + 17 2
  15 + 13 2
  5 + 12 5
  Vehicle + 16 4
  TA 1537 5000 - 6 4
  1500 - 6 2
  500 - 7 3
  150 - 11 3
  50 - 7 2
  15 - 6 2
  5 - 11 5
  Vehicle - 10 2
  5000 + 8 1
  1500 + 11 4
  500 + 11 2
  150 + 12 4
  50 + 10 4
  15 + 12 3
  5 + 9 3
  Vehicle + 14 4
  TA 98 5000 - 23 2
  1500 - 24 2
  500 - 20 2
  150 - 25 7
  50 - 19 2
  15 - 22 7
  5 - 25 1
  Vehicle - 25 1
  5000 + 30 1
  1500 + 26 3
  500 + 25 1
  150 + 27 9
  50 + 26 4
  15 + 27 3
  5 + 24 5
  Vehicle + 30 5
  TA 100 5000 - 92 9
  1500 - 112 15
  500 - 98 8
  150 - 98 3
  50 - 98 20
  15 - 119 17
  5 - 123 5
  Vehicle - 113 7
  5000 + 76 2
  1500 + 108 4
  500 + 111 10
  150 + 117 15
  50 + 115 21
  15 + 118 16
  5 + 97 11
  Vehicle + 114 6
  CM 891 5000 - 181 17
  1500 - 242 4
  500 - 194 19
  150 - 206 7
  50 - 220 19
  15 - 238 12
  5 - 213 3
  Vehicle - 235 22
  5000 + 215 30
  1500 + 228 9
  500 + 189 19
  150 + 209 5
  50 + 203 4
  15 + 210 45
  5 + 210 5
  Vehicle + 221 12
2 TA 1535 5000 - 15 2
  1500 - 18 2
  500 - 12 2
  150 - 15 4
  50 - 9 1
  Vehicle - 14 1
  5000 + 14 0
  1500 + 14 5
  500 + 12 2
  150 + 14 1
  50 + 14 1
  Vehicle + 14 3
  TA 1537 5000 - 11 2
  1500 - 10 4
  500 - 10 3
  150 - 8 1
  50 - 10 2
  Vehicle - 10 1
  5000 + 11 3
  1500 + 11 4
  500 + 9 2
  150 + 13 2
  50 + 13 2
  Vehicle + 12 2
  TA 98 5000 - 27 6
  1500 - 23 3
  500 - 23 4
  150 - 24 3
  50 - 20 10
  Vehicle - 24 2
  5000 + 26 4
  1500 + 24 2
  500 + 27 2
  150 + 28 4
  50 + 27 2
  Vehicle + 29 6
  TA 100 5000 - 113 6
  1500 - 115 16
  500 - 115 7
  150 - 111 13
  50 - 110 13
  Vehicle - 107 7
  5000 + 111 4
  1500 + 124 14
  500 + 122 14
  150 + 115 9
  50 + 117 11
  Vehicle + 109 9
  CM 891 5000 - 190 19
  1500 - 186 6
  500 - 199 5
  150 - 211 9
  50 - 195 9
  Vehicle - 189 5
  5000 + 198 10
  1500 + 209 11
  500 + 224 8
  150 + 217 14
  50 + 200 6
    Vehicle + 193 13

CM 891 - WP2uvrA/pKM101

-      absence 

+   presence

SD  Standard deviation

Table 3. Revertant colony counts per plate for positive controls

Test Strain Compound Concentration S9 mix Mean revertant colony counts SD
      (µg/plate)    
1 TA 1535 NaAz 0.50 - 182 34
  TA 1537 9AC 30.00 - 71 6
  TA 98 NF 1.00 - 211 12
  TA 100 NaAz 0.50 - 402 17
  CM 891 AF-2 0.05 - 2006 22
  TA 1535 AA 2.00 + 122 6
  TA 1537 B[a]P 5.00 + 69 6
  TA 98 B[a]P 5.00 + 229 17
  TA 100 B[a]P 5.00 + 750 61
  CM 891 AA 10.00 + 1848 38
2 TA 1535 NaAz 0.50 - 296 25
  TA 1537 9AC 30.00 - 125 4
  TA 98 NF 1.00 - 180 21
  TA 100 NaAz 0.50 - 541 9
  CM 891 AF-2 0.05 - 2365 35
  TA 1535 AA 2.00 + 101 11
  TA 1537 B[a]P 5.00 + 108 11
  TA 98 B[a]P 5.00 + 337 19
  TA 100 B[a]P 5.00 + 856 28
  CM 891 AA 10.00 + 1836 16

CM 891 - WP2uvrA/pKM101

-       absence 

+   presence

SD  Standard deviation

NaAz: sodium azide

9AC: 9-aminoacridine

NF: 2-nitrofluorene

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

AA: 2-aminoanthracene

B[a]P: benzo[a]pyrene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the test, the test material shows no evidence of mutagenic activity in the bacterial system and therefore is considered to be negative for genotoxicity.
Executive summary:

In a GLP bacterial reverse mutation assay conducted in accordance with standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100, the genotoxicity of the test substance was determined. Four strains of S. typhimurium and one strain of E. coli were treated in the absence and presence of metabolic activation. Under the conditions of the test, no evidence of mutagenic activity in the bacterial system was observed and the test material was determined to be negative for genotoxicity.