Ammonium nitrate

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:

September 05-18, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was performed with a substance analogue and the data are read across.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium nitrate

EC Number:

233-332-1

EC Name:

Calcium nitrate

Cas Number:

10124-37-5

Molecular formula:

Ca(NO3)2

IUPAC Name:

calcium dinitrate

Constituent 2

Reference substance name:

Ammonium nitrate

EC Number:

229-347-8

EC Name:

Ammonium nitrate

Cas Number:

6484-52-2

Molecular formula:

H3N.HNO3

IUPAC Name:

ammonium nitrate

Constituent 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid: granular

Details on test material:

- Name of test material (as cited in study report): CN-nitcal
- Physical appearance: white granules
- Storage conditions:

Method

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in E. coli

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:

phenobarbital and beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
first and second test: 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: at concentrations of 33.3 mg/ml and lower the test substance was dissolved in the vehicle.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix Migrated to IUCLID6: 5 μg/plate in saline for TA1535

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 60 µg/plate in mili-Q water for TA1537

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Controls 4

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO (5 and/or 10%) for all tester strains

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicates in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the relevant colonies


OTHER EXAMINATIONS:
- Other: precipitation of the test substance

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent
control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater
than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or
the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3)
times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one
independently repeated experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance did not precipitate at the start or at the end of the incubation period


RANGE-FINDING/SCREENING STUDIES:
No toxicity and mutagenicity was observed up to concentrations of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data
ranges indicating that the test conditions were adequate and that the metabolic activation system functioned
properly.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metobolic activation system functioned properly.
Based on the results of this study it is concluded that CN-Nitcal is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.