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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:
Oct. 6, 2004 to Dec. 19, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 471) with acceptable restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
- 2-anthramine was the only positive control compound used to test the efficacy of the S9 fraction
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1H-1,2,4-triazole, sodium salt
EC Number:
255-280-9
EC Name:
1H-1,2,4-triazole, sodium salt
Cas Number:
41253-21-8
Molecular formula:
C2H3N3.Na
IUPAC Name:
sodium 1,2,4-triazol-1-ide
Details on test material:
- Name of test material (as cited in study report): 1,2,4-Triazole, Sodium salt
- Physical state: White powder
- Analytical purity: 99.3%
- Lot/batch No.: 0552
- Expiration date of the lot/batch: 30 September 2005

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver microsomes
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500, 3750 and 5000 µg/plate
Vehicle / solvent:
The vehicle was water for injections, batch No. FVCOIA (Frésenius-Kabi, Sèvres, France).
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See Table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): revertants were scored with an automatic counter
- Fixation time (start of exposure up to fixation or harvest of cells): no data

NUMBER OF REPLICATIONS: 2 independent experiments, 3 plates/dose
NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: no data on used method

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no

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:
cytotoxicity
Remarks:
Moderate-to-marked toxicity at > 2500 µg/plate (+/- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Moderate-to-marked toxicity at > 2500 µg/plate (+/- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
A moderate to marked toxicity was generally observed, both with and without S9 mix, mainly at dose-levels = 2500 µg/plate, depending on the tester strains. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.

Applicant's summary and conclusion

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

Under the experimental conditions, 1,2,4 -triazole sodium salt did not show any mutagenic activity in the bacterial reverse mutation test.
Executive summary:

The study was performed according to the international guidelines (OECD 471, Commission Directive No. B 13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels of 1,2,4-triazole sodium salt to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Five strains of bacteria Salmonella Typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. 1,2,4-triazole sodium saltwas dissolved in water for injections.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

The selected treatment-levels were: 312.5, 625, 1250, 2500, 3750 and 5000 gg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.

A moderate to marked toxicity was generally observed, both with and without S9 mix, mainly at dose-levels 2500 gg/plate, depending on the tester strains.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.