Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2000-11-09 to 2001-02-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Tantalum pentoxide used as read-across partner.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Ta2O5 Tantalum Pentoxide Grade LT
- Molecular formula (if other than submission substance): Ta2O5
- Physical state: white powder
- Analytical purity: 99.99%
- Lot/batch No.: 991165
- Storage condition of test material: room temperature, in the dark under dry conditions

Method

Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
First test (range-finding): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Second test: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
Purified water containing 0.15 % agar.
The test substance was found to be insufficiently soluble in all compatible solvents. Therefore, it was suspended in purified water (obtained by reverse-osmosis) containing 0.15 % agar (prepared in-house).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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, 2-aminoanthracene
Details on test system and experimental conditions:
MUTATION TEST PROCEDURE
First test (range-finding)
The test substance was added to cultures of the five tester strains at seven concentrations separated by ca. half-log10 intervals. The highest concentration of Ta2O5 Tantalum Pentoxide Grade LT tested was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 µg/plate. The negative control was the chosen vehicle, purified water containing 0.15% agar. The appropriate positive controls were also included.

Protocol:
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S9 mix or 0.5 mL 0.1 M sodium phosphate buffer (pH 7.4) were placed in glass tubes. An aliquot of 0.1 mL of the test dilution, positive or negative control was added, followed immediately by 2 mL of molten agar containing 0.5mM histidine/biotin/tryptophan. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish's contents. Three petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37 °C for ca 72 hours. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. 5000 µg/plate was again chosen as the top concentration, but only five concentrations were used.
Evaluation criteria:
ASSESSMENT OF RESULTS
For a test to be considered valid the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within the 99 % confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a minimum of two and maximum of five years. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.

The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, the test substance will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test, the test substance will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a) and b), even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett's test.
Statistics:
Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocalresponse if no clear results can be obtained.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
RESULTS
The absence of colonies on sterility check plates confirmed the absence of microbial contamination.
The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory (except strain TA98, test 2, where counts were slightly higher; this was not considered to affect the integrity of the study). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.
FIRST TEST (RANGE-FINDING)
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Ta205 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to Ta205. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
SECOND TEST
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Ta205 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to Ta205.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, the test substance Tantalum Pentoxide Grade LT showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic potential of the test substance in accordance to OECD guideline 471, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to the test substance diluted in purified water containing 0.15% agar. Purified water containing 0.15% agar was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay; the second involved a pre-incubation stage.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions, the test substance showed no evidence of mutagenic activity in this bacterial system. Ta2O5 Tantalum Pentoxide Grade LT is used as read-across partner to tantalum pentachloride.