N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

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:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

- Only four (4) strains were used instead of five (5).
- The selected S.typhimurium strains did not include the one (T102) that is known to be particularly susceptible to certain oxidizing or cross-linking agents.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

EC Number:

266-257-8

EC Name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Cas Number:

66215-27-8

Molecular formula:

C6H10N6

IUPAC Name:

N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Test material form:

solid: particulate/powder

Remarks:

white

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: Nine concentrations ranging from 0.08 to 5000 µg/plate.
Bacterial Mutation assay: Two trials were carried out using S. typhimurium (TA 1535, TA 1537, TA 100, TA 98) strains. For both trials with or without metabolic activation concentrations of 0, 20, 78, 313, 1250 and 5000 µg cyromazine/plate were used.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other:

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cyclophosphamide

other:

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Bacterial mutation assay: 0.1 mL of appropriate positive and negative controls or test article (cyromazine) solutions, 0.1 mL of the bacterial tester strain, 2.0 mL of the prediluted Ames top agar (45°C) and 0.5 mL of S9 mix for the metabolic activation method were put in a tube mixed and poured onto a plate containing the base agar medium. The top agar layer was allowed to gel at room temperature and then plates (triplicate in each test point) inverted and incubated at 37°C for 48 hours. Revertant colonies per plate were counted using an automatic counter.

Rationale for test conditions:

Nine concentrations ranging from 0.08 to 5000 µg/plate were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained (see below), the highest concentration suitable for the mutagenicity test was found to be 5000 µg/plate.

The concentration in the stock solutions were confirmed analytically by Gas Liquid Chromatography using a phosphorus/nitrogen detector.

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537,
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration related effect should be demonstrable.

Results and discussion

Test results

Key result

Species / strain:

other: All strains used in this study

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:

The content of Cyromazine in the stock solutions were found to be 104.4 and 94.8% in the first and second experiment, respectively.

Any other information on results incl. tables

See overall remarks attachments

Applicant's summary and conclusion

Conclusions:

Cyromazine was not mutagenic in a bacterial reverse mutation assay at concentrations up to 5000 µg/plate, in the absence or presence of metabolic activation system, respectively.

Executive summary:

Cyromazine was tested for mutagenic effects on histidineauxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and 1537 without and with microsomal activation with the following concentrations of the trial substance: 20, 78, 313, 1250 and 5000 µg/plate. In order to confirm the results the experiments were repeated.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat-liver microsomes and co-factors). The test material in solution was analysed to confirm the intended concentrations to be used in the mutagenicity tests. The values found were in agreement with the calculated concentrations.

In the experiments performed without and with microsomal activation, none of the tested concentrations of cyromazine led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by Cyromazine or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.