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Genetic toxicity: in vitro

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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:
2001-03-21 to 2001-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 1999-01-25
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Boron
EC Number:
231-151-2
EC Name:
Boron
Cas Number:
7440-42-8
Molecular formula:
B
IUPAC Name:
Boron
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Boron amorphous
- Physical state: brown powder
- Storage condition of test material: room temperature, dry
- Average particle size: 0.9 µm
- Specific surface area: 11.1 m^2/g

Method

Target gene:
not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
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 (pre-incubation assay): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water containing 0.15% agar (prepared in-house)
- Justification for choice of solvent/vehicle: the test substance was insoluble in all compatible solvents at 50 mg/mL. Suspensions of the test substance were, therefore, prepared in purified water containing 0.15% agar (prepared in-house).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation; strains TA1535 (0.5 µg/plate) and TA100 (0.5 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Positive control without metabolic activation; strain TA1537 (30 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Positive control without metabolic activation; strain TA98 (1µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
Positive control without metabolic activation; strain WP2uvrA/pKM101 (CM891) (0.05 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 - Aminoanthracene
Remarks:
Positive control with metabolic activation; strain WP2uvrA/pKM101 (CM891) (10 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive control with metabolic activation; strains TA1537 (5 µg/plate), TA98 (5 µg/plate) and TA100 (5 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)(first test) and preincubation (second test)

NUMBER OF REPLICATIONS: three petri dishes were used for each concentration

NUMBER OF CELLS EVALUATED: the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

CHECK FOR STERILITY:
Plates were prepared without addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer.

VALIDITY OF TEST
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. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.

Evaluation criteria:
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 consideredto 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. Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

*Reference:
Mahon, G. A. T. et al. (1989) Analysis of data from microbial colony assays in Kirkland, D. J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, pp. 26 - 65. Cambridge University Press, Cambridge.
Statistics:
Please refer to "Evaluation criteria" above

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on 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 vehicle controls were within the 99% confidence limits of the current historical control range of the laboratory. 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.

RESULTS FIRST TEST
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to boron amorphous 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 boron amorphous. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.

RESULTS SECOND TEST
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to boron amorphous 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 boron amorphous.
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

It is concluded that, under the test conditions employed, boron amorphous showed no evidence of mutagenic activity in this bacterial system.

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