2H-Tetrazol-5-amine, potassium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

potassium 5-amino-1H-1,2,3,4-tetrazol-1-ide

Cas Number:

136369-04-5

Molecular formula:

CH2KN5

IUPAC Name:

potassium 5-amino-1H-1,2,3,4-tetrazol-1-ide

Test material form:

solid: particulate/powder

Details on test material:

Batch no.: 08KI030

Method

Target gene:

TA97a: hisD6610, uvrB, pKM 101, rfa
TA 98: hisD3052, uvrB, pKM 101, rfa
TA 100 : hisG46, uvrB, pKM 101, rfa
TA102: hisG428, pKM 101, rfa
TA1535 : hisG46, uvrB, rfa.

Species / strain

Species / strain / cell type:

other: S. typhimurium LT2, TA 1535, TA 97a, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 Mix

Test concentrations with justification for top dose:

50 µg/plate, 151 µg/plate, 502 µg/plate, 1505 µg/plate, 5015 µg/plate,

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

7.1 Culture of Bacteria
10 hours before the start of each experiment, a stock culture of each strain was thawed and an aliquot was placed into a culture flask containing 70 mL nutrient broth. The flasks were incubated at 37°C for 10 hours.

7.2 Description of the Method
7.2.1 Preparations In the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilized and the first batches poured. On the day before the test, the remaining plates were poured. On the day of the test, the overnight cultures were checked for growth. The incubation chambers were heated to 37°C. The water bath was turned to 43 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction. A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor  2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium LT2, TA 1535, TA 97a, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item didn’t show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item 5-Aminotetrazol Potassium is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

The test item 5-Aminotetrazol Potassium is considered as “not mutagenic under the conditions of the test”.

Executive summary:

First Experiment:

Five concentrations of the test item, dissolved in deionised water (ranging from 5015 to 50 μg/plate) were used. Five genetically manipulated strains of Salmonella typhimurium (TA

97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the

plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn’t show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The

determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic

activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging from 4999 to 1250 μg/plate) and a modification

in study performance (pre-incubation method). The test item didn’t show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Under the conditions of the test, the test item didn’t show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Therefore, no concentration-effect relationship could be determined.