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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.10.2017 – 12.01.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrazol-5-ylamine
EC Number:
224-581-7
EC Name:
Tetrazol-5-ylamine
Cas Number:
4418-61-5
Molecular formula:
CH3N5
IUPAC Name:
1H-tetrazol-5-amine
impurity 1
Reference substance name:
Unknown impurities
Molecular formula:
Not available
IUPAC Name:
Unknown impurities
Test material form:
solid: crystalline
Details on test material:
white crystals

Method

Target gene:
gene for histidine or tryptophan synthesis
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:
supernatant of rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 μg
Selection of doses/toxicity:
Sponsor declared good solubility of the test substance in water. It was not confirmed – after mixture with water in concentration demanded for testing (5000 µg per 0.1 mL) most of the test substance remained undissolved.
Good solubility was found in dimethyl sulfoxide where the test substance melted in immediately in the same concentration.
For toxicity experiment the highest concentration 5 mg per mL (recommended in OECD TG 471) was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 100 without metabolic activation
No precipitation was observed in top agar or in Petri dishes.
Thinner bacterial background was observed in the highest concentration. As toxicity in the highest dose is allowed, the concentration of 5000 µg per plate was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.

In the first mutagenicity experiments, the highest concentration was mostly toxic for S. typhimurium TA 98, where revertants could not be counted due to colonies in background. In the other bacterial strains, thinner bacterial background occured in the highest concentration.

In the second mutagenicity experiments concentrations were decreased. The maximum concentration 3000 µg per plate was diluted analogically as in the first experiments because of no signs of mutagenicity were noticed. To increase the sensitivity of the assay volume of S9 was increased from 30 to 50 µL.
Fresh suspensions/solutions of the test substance were prepared before each experiment. Concentrations of the test substance suspension/solution were dosed in the volume of 0.1 mL per plate. In all experiments, tubes with application forms of the test substance as well as tubes with top agar were shaken before any handling.
Vehicle / solvent:
dimethyl sulfoxide (DMSO), Merck Lot. No. K48069252 644, exp. 10/2018
- Justification for choice of solvent/vehicle: solubility of the substance
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
negative controls - 0.1 mL of DMSO
Remarks:
Untreated controls - no solvent
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
(AS)
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
(NPD)
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Remarks:
(2-AF)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
(2-AA)
Positive controls:
yes
Positive control substance:
other: N-methyl-N´-nitro-N-nitrosoguanidine
Remarks:
(MNNG)
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Remarks:
(9-AAc)
Details on test system and experimental conditions:
The bacterial tester strains
Salmonella typhimurium
TA 1535 (CCM 3814, lot. No. 2101200916917),
TA 98 (CCM 3811, lot No. 01022001220053),
TA 100 (CCM 3812, lot No. 0102201220054) and
TA 1537 (CCM 3815, lot No. 2101200916918)
as well as
Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732),
were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2uvrA detects cross-linking mutagens


METHOD OF APPLICATION: in medium; in agar (plate incorporation)

NUMBER OF REPLICATIONS: two series

DETERMINATION OF CYTOTOXICITY
- Method: decrease of number of revertants or diminution of bacterial background

Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (see below). After this rule the result is positive, if a reproducible doseresponse effect occurs and/or a doubling of the ratio Rt/Rc is reached.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods:
Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation. Elsevier North-Holland Biomedical Press. 231 - 417
Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189. 83 - 91

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity in the highest dose is allowed
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative 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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of DMSO. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Sponsor declared good solubility of the test substance in water. It was not confirmed – after mixture with water in concentration demanded for testing (5000 µg per 0.1 mL) most of the test substance remained undissolved. Good solubility was found in dimethyl sulfoxide where the test substance melted in immediately in the same concentration.
For toxicity experiment the highest concentration 5 mg per mL (recommended in OECD TG 471) was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 100 without metabolic activation
No precipitation was observed in top agar or in Petri dishes.
Thinner bacterial background was observed in the highest concentration. As toxicity in the highest dose is allowed, the concentration of 5000 µg per plate was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.

In preliminary cytotoxicity test in Salmonella typhimurium TA 100 without metabolic activation, toxicity occurred in the highest concentration, which was observed as partial diminution of background.
In the first mutagenicity experiment the test substance in the highest concentration was mostly toxic for S. typhimurium TA 98, where revertants could not be counted due to colonies in background. In the other bacterial strains toxicity was manifested as diminution of bacterial background.
In the second mutagenicity experiment, concentrations were generally decreased. Diminution of bacterial background was observed in some bacterial strain in the highest concentration as well as decreased number of revertants (S. typhimurium TA 1535, TA 1537 and TA 98 without metabolic activation).
Remarks on result:
other: toxicity was observed as partial diminution of background

Applicant's summary and conclusion

Conclusions:
In the arrangement given above, the test substance, 5-aminotetrazole, was non-mutagenic for all the used tester strains without as well as with metabolic activation.
Executive summary:

The test substance, 5-aminotetrazole, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 30-5000 μg per plate, which were applied to plates in volume of 0.1 mL.

The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL S9 per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50 – 5000 μg per plate.

The highest concentration was partially toxic, so lower concentrations (30-3000 μg per plate) were used and volume of S9 was increased to 50 μL per plate.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the above-described experimental design, the test substance, 5-aminotetrazole, was non-mutagenic for all the used tester strains without as well as with metabolic activation.