1-nitroguanidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2013 - 02 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1-nitroguanidine

EC Number:

209-143-5

EC Name:

1-nitroguanidine

Cas Number:

556-88-7

Molecular formula:

CH4N4O2

IUPAC Name:

N-nitroguanidine

impurity 1

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

Na2O4S

IUPAC Name:

disodium sulfate

impurity 2

Reference substance name:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

impurity 3

Reference substance name:

4,6-diamino-1,3,5-triazin-2(1H)-one

EC Number:

211-455-1

EC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

Cas Number:

645-92-1

Molecular formula:

C3H5N5O

IUPAC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

impurity 4

Reference substance name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

EC Number:

211-456-7

EC Name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

Cas Number:

645-93-2

Molecular formula:

C3H4N4O2

IUPAC Name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

additive 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test substance supplier NIgu Chemie GmbH
Los 3286

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

- collected from a single donor who was a healthy, non-smoking individual with no kown recent exposures to pharmaceuticals, genotoxic chemicals, or radiation
- drawn by venous puncture and collected in heparinized tubes
- blood was stored under sterile conditions at 4 derees C for a maximium of 4 hours
- whole blood samples treated with an anti-coagulant were pre-cultured in the presence of mitogen

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

without: EMS; with: CPA

Test concentrations with justification for top dose:

-Experiment 1: without metabolic activation - 15, 20, 180, 360, 750, 1250, 2500, 3000, 4000, 5000 ug/mL
-Experiment 1: with metabolic activation - 180, 360, 750, 1250, 2500, 3000, 3500, 4000, 5000 ug/mL
-Experiment 2: without metabolic activation - 750, 1250, 2500, 3000, 3500, 4000, 4500, 5000 ug/mL
-Experiment 2: with metabolic activation - 500, 1000, 2000, 3500, 4500, 5000 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI + 0 % fetal bovine serum
- Justification for choice of solvent/vehicle: based on the results of the solubility test cell culture medium cell culture medium was used as a solvent

Controls

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Experiment 1: without and with metabolic activation, 4h treatment, 24h preparation interval - 1250, 2500, 5000 ug/mL
- Experiment 2: without metabolic activation, 24h treatment, 24h preparation interval - 1250, 2500, 5000 ug/mL
- Experiment 2: with metabolic activation, 4h treatment, 24h preparation interval - 1000, 2000, 5000 ug/mL
- at least 3 analysable concentratiosn of hte test item were used for the 24 h preparation

DETERMINATION OF CYTOTOXICITY
- Method: proliferation index, mitotic index

Evaluation criteria:

The chromosomal aberration assay is considered acceptable if:
- The number of aberration found in the negative and/or solvent controls fall within the range of historical laboratory control data: 0-4 % (without and with metabolic activation)
- The positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations

Criteria for determining a positive result:
- A clear and dose-related increase in the number of cells with aberrations
- A biologically relevant response for at least one dose group, which is higher than the laboratory negative control range (0-4 % without and with metabolic activation)

Statistics:

- Statistical significance (P < 0.05) was determined by Fischer's exact test

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test substance precipitates at 2 and 5 mg, therefore there is no genotoxicity up to the maximum solubility.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment 2, with and without metabolic activation, a biologically relevant decrease of the relative mitotic index was seen at 5000 ug/mL.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Nitroguanidine did not induce structural chromosomal aberrations in human lymphocytes and is therefore considered non-clastogenic. Furthermore, the test substance precipitates at 2 and 5 mg, therefore there is no genotoxicity up to the maximum solubility.

Executive summary:

In a mammalian cell cytogenetics chromosomal aberration assay primary human lymphocyte cultures were exposed to nitroguanidine at the following concentrations:

-Experiment 1: without metabolic activation - 15, 20, 180, 360, 750, 1250, 2500, 3000, 4000, 5000 µg/mL

-Experiment 1: with metabolic activation - 180, 360, 750, 1250, 2500, 3000, 3500, 4000, 5000 µg/mL

-Experiment 2: without metabolic activation - 750, 1250, 2500, 3000, 3500, 4000, 4500, 5000 µg/mL

-Experiment 2: with metabolic activation - 500, 1000, 2000, 3500, 4500, 5000 µg/mL 

Nitroguanidine was tested up to 5000 µg/mL because the guideline recommends a maximum concentration of 5 mg/mL. Positive controls induced the appropriate response. There was no evidence of chromosomal aberration induced over background.

This study is classified as acceptable.  This study satisfies the requirement of test guidelines OECD 473 (in vitro mammalian chromosome aberration test), EU method B.10 (mutagenicity - in vitro mammalian chromosome aberration test), and EPA OPPTS 870.5375 - in vitro mammalian chromosome aberration test for in vitro cytogenetic mutagenicity data.