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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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

Constituent 1
Chemical structure
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
Chemical structure
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
Chemical structure
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix; 5 μg/plate in saline for TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix; 60 µg/plate in mili-Q water for TA1537
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9-mix; 10 µg/plate in DMSO for TA98
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix; 650 µg/plate in DMSO for TA100
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 µg/plate in DMSO for WP2uvrA
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 resultsopen allclose all
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
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.