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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:
From 26 August to 17 October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material : 3-NITRO-1 ,2,4-TRIAZOL-5-0NE (NTO)
- Substance type: energetic explosive
- Physical state: light green to white crystalline solid with no odor
- Purity ca.99%
Specific details on test material used for the study:
Supplied by Ordnance Systems, Inc., Kingsport, Tennessee
Lot no.:BAE 07B 305001
Purity:99.6%

Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
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
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range finding test: 5, 10, 50, 100, 500, 1000 and 5000 μg/plate
Mutation test: 5, 10, 50, 100 and 250 μg/plate for Salmonella typhimurium and 100, 250, 500, 750 and 1000 μg/plate for Escherichia coli without activation. With activation, the dose levels were 100, 500, 1000, 2500 and 5000 μg/plate for both Salmonella typhimurium and Escherichia coli.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility in H2O was only 14.28 mg/mL. It was soluble in DMSO at about 500 mg/mL and formed a clear yellow solution.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
In the absence of S9-mix for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
In the absence of S9-mix for strains TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In the absence of S9-mix for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
In the absence of S9-mix for strain WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the presence of S9-mix for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48 to 72 hours
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 48 to 72 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 250-500 cells

OTHER EXAMINATIONS:
- Other: After the incubation period was completed, the plates, starting with the highest test article concentration, were observed for the presence of precipitate. Plates were counted for the frequency of revertant colonies using an ARTEK counter, model 880. Three counts were taken by rotating the plate on the counter stage and the median count was entered into a validated, MS Office Excel spreadsheet program designated as "2140B.xlw". The background lawn was also evaluated. The same notations as in the Range Finding Test were used to evaluate the precipitate and background lawn.

OTHER: In order to determine the toxicity of the test article and to select appropriate test article concentrations for the Definitive Mutation Assay, a Range Finding Test was performed using strains TA100 and WP2 uvrA.
To confirm the results of the Definitive Assay, Confirmatory Mutation Assays were performed using the plate incorporation method at test article concentrations of 10, 50, 100, 250 and 500 μg/plate for Salmonella typhimurium and 250, 500, 750, 1000 and 2500 μg/plate for Escherichia coli without activation. With activation, the dose levels were 100, 500, 1000, 2500 and 5000 μg/plate for both Salmonella typhimurium and Escherichia coli. All test article concentrations, including the controls, were tested in triplicate.

No additional data
Evaluation criteria:
Criteria for a Negative Response:
A response was considered to be negative if all of the strains treated with the test article had mean reversion frequencies that were less than twice that of the mean reversion frequencies of the corresponding solvent control plates in TA98 and TA100 and less than three times in TA1535, TA1537 and WP2 uvrA, and there was no evidence of a concentration-dependent response.

Criteria for a Positive Response:
A response was considered to be positive if either strain TA98 or TA100 exhibited a mean reversion frequency that was at least double the mean reversion frequency of the corresponding solvent control in at least one concentration, or if either strain TA1535, TA1537 or WP2 uvrA exhibited a three-fold increase in the mean reversion frequency compared to the solvent control in at least one concentration. In addition, the response must have been concentration-dependent or increasing concentrations of the test article must have showed increasing mean reversion frequencies. In evaluating the results, consideration was given to the degree of toxicity exhibited by the concentration causing the 2 to 3-fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency.

Criteria for an Equivocal Response:
A response was considered equivocal if it did not fulfill the criteria of either a negative or a positive response and/or the Study Director did not consider the response to be either positive or negative.
Statistics:
None stated

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
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range finding test:
TA100: The Relative Cloning Efficiencies (RCEs) at the concentrations of 5.0 to 5000 μg/plate without activation were from 2% to 87%. The revertants were significantly decreased at 500 μg/plate and above and the background lawns were absent at 5000 μg/plate. In the activation system, the RCEs at the concentrations of 5.0 to 5000 μg/plate were 37% to 109%. The significant decreased RCE (<50%) was only found at 5000 μg/plate. The revertants were not significantly decreased at any dose levels. No precipitate was observed at any of the test concentrations.
WP2uvrA: The Relative Cloning Efficiencies (RCEs) at the concentrations of 5.0 to 5000 μg/plate without activation were from 1 % to 155%. The RCEs were 16% at 1 000 μg/plate and 1 % at 5000 μg/plate. The revertants were significantly decreased at 5000 μg/plate and the background lawn was also absent. In the activation system, the RCEs were from 55% to 110% at the concentrations from 5.0 to 5000 μg/plate. The revertants were significantly decreased only at 5000 μg/plate. No precipitate was observed at any of the test concentrations.

Definitive Mutation Assay:
The revertants from all of the test article-treated plates for all strains were not significantly greater than those of their corresponding solvent controls. The background lawns were normal for all concentrations. Based on these results, the Definitive Mutation Assay was negative. Both the solvent and positive controls fulfilled the requirements of the test.

Confirmatory Mutation Assay:
As in the Definitive Assay all strains treated with test article had revertant counts that were not significantly greater than those of their corresponding solvent controls. The background lawns were normal at all concentrations tested. Therefore, the results were negative. The solvent and positive controls for all data presented fulfilled the requirements of the test.
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

Under the conditions of this study, the test article was negative in the Salmonella typhimurium/Escherichia coli Plate Incorporation Mutation Assay both with and without activation.