Sodium nitrate

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:

12-mar-2010 to 25-apr-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Natriumnitrat HQ unbehandelt (non-food grade)
- Substance type: White crystalline powder
- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 10, 33, 100, 333 and 850 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 10, 33, 100, 333 and 850 µg/mL
First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 100, 333 and 850 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 333 and 850 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 100, 333 and 850 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 100, 333 and 850 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: exposure medium (RPMI 1640 medium )
- Justification for choice of solvent/vehicle: Test compound was soluble in exposure medium and exposure medium has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9; in Hank's Balanced Salt Solution: 0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period

Controls 2

Positive control substance:

cyclophosphamide

Remarks:

with S9; in Hank's Balanced Salt Solution: 10 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood

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:

- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 850 µg/mL (= 0.01 M)
RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 850 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 850 µg/mL

Any other information on results incl. tables

No effects of Natriumnitrat HQ unbehandelt (non-food grade) on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Natriumnitrat HQ unbehandelt (non-food grade) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations

Applicant's summary and conclusion

Conclusions:

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Natriumnitrat HQ unbehandelt (non-food grade) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.


Finally, it is concluded that this test is valid and that Natriumnitrat HQ unbehandelt (non-food grade) is not clastogenic in human lymphocytes under the experimental conditions described in the report.