4H-1,2,4-triazol-4-ylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.06.2021 - 06.08.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted June 26, 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4H-1,2,4-triazol-4-ylamine

EC Number:

209-533-5

EC Name:

4H-1,2,4-triazol-4-ylamine

Cas Number:

584-13-4

Molecular formula:

C2H4N4

IUPAC Name:

4H-1,2,4-triazol-4-ylamine

Test material form:

solid

Method

Target gene:

gene for histidine or tryptophan synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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:

1st: 50, 150, 500, 1500 and 5000 μg
2nd: 50, 150, 500, 1500 and 5000 μg
3rd: 1000, 2000, 3000, 4000 and 5000 μg

Selection of doses/toxicity:
Based on the cytotoxicity experiment, the first mutagenicity experiments were performed as plate incorporation test with and without metabolic activation in all the strains.
The concentration of 5000 μg per plate was used as maximum. Further concentrations were obtained by diluting the maximum concentration. The dilution factor used was between 2 and √10, with resulting concentration range 50, 150, 500, 1500 and 5000 ug per plate.

No precipitation, cytotoxicity and no signs of mutagenicity were observed in the first mutagenicity experiments, so the results of the first mutagenicity experiments were evaluated as non mutagenic for all the bacterial strains used.

In case of negative results, the OECD TG 471 requires either reasonable justification of no further testing or the confirmation of negative experiment with another test with changes in experiment arrangement. So, we decided to perform the second mutagenicity experiments with pre-incubation for increase of sensitivity. Based on the previous experiments, cytotoxicity was not expected, so the same concentrations were then used in the second mutagenicity experiments.

No cytotoxicity and some increased numbers of revertants in S. typhimurium TA 100 and TA 1535 were observed in the experiments with pre-incubation and metabolic activation, so these results were examined in the third mutagenicity experiment conducted in the same manner as the second ones with concentrations of 1000, 2000, 3000, 4000 and 5000 ug per plate.

Vehicle / solvent:

water for injection: Ardeapharma 1909120570, exp. 09/2021

- Justification for choice of solvent/vehicle: solubility of the substance
The test item is soluble in water a solution of in water for injection was prepared in the maximum recommended concentration 5000 μg per plate/0.1 mL.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

confirms non-toxicity, non-mutagenicity and sterility of vehiculum

Controls 2

Untreated negative controls:

yes

Remarks:

no solvent, confirms one of properties (spontaneous reversion) of bacterial strain

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(AS)

Controls 4

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

(NPD)

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Remarks:

(2-AF)

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2-AA)

Controls 7

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Remarks:

(MNNG)

Controls 8

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 Cat. No. 3814, lot. No. 2101200916917,
TA 98, Cat. No. CCM 3811, lot No. 0102201220053 and
TA 100 Cat. No.CCM 3812, lot No. 0102201220054
were obtained from Czech Collection of Microorganisms (CCM), Brno (CZ).

TA 1537 comes from Xenometrix Cat. No. PSS-0113, lot No. 18d

Escherichia coli WP2 uvrA was obtained from Xenometrix (lot no. U20),

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

Genotypes of strains: Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).


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

NUMBER OF REPLICATIONS: two series

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods (see below). Per 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 results

Test results 1

Key result

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

Test results 2

Key result

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

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

Test results 5

Key result

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 see Chapter 4. Study Results), negative and solvent controls. Negative controls contain no solvent, and solvent controls contain 0.1 of solvent (water for injection). 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:
The cytotoxicity experiment was performed as plate incorporation test for determination of concentrations in the first mutagenicity experiment in the same way as mutagenicity experiments in Salmonella typhimurium TA98, experiment without metabolic activation using two plates per concentration.
The highest concentration recommended according to the OECD TG 471, 5 mg per plate, was further diluted with a factor 2-√3. The range of concentrations was 10, 100, 500, 1000, 2500 and 5000 μg per plate.
Precipitation or cytotoxicity were not observed in any concentration.

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test item, 4H-1,2,4-triazol-4-amine, was found to be non mutagenic for all the used indicator strains with and without metabolic activation.

Executive summary:

The test item, 4H-1,2,4-triazol-4-amine, was assayed for mutagenicity using the Bacterial Reverse Mutation Test. The test was performed according to the OECD Test Guideline No. 471 in agreement with the EU Method B.13/14.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used.

As the test item is soluble in water a solution of in water for injection was prepared in the maximum recommended concentration 5000 μg per plate/0.1 mL.  A cytotoxicity experiment was performed first, with a concentration range of 10-5000 μg per plate in Salmonella typhimurium TA98 without metabolic activation. Based on the results, the concentrations used in the first mutagenicity experiments (plate incorporation) were 50, 150, 500, 1500 and 5000 μg per plate.

No cytotoxicity, no precipitation and no mutagenicity were observed in the first mutagenicity experiments. Therefore, the second mutagenicity experiment was performed with pre-incubation and with the same concentrations as the first one.

Some increased number of revertants were observed in the second mutagenicity experiments with metabolic activation, so the third mutagenicity experiment was performed in these bacterial strains, with preincubation, with metabolic activation and the test item concentrations of 1000, 2000, 3000, 4000, 5000. Mutagenicity was not confirmed.

 

In the arrangement given above, the test item, 4H-1,2,4-triazol-4-amine, was found to be non‑mutagenic for all the used indicator strains with and without metabolic activation.