Sodium nitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium nitrate is not mutagenic in Ames and chromosome aberration studies with human lymphocytes with and without metabolic activation. Several less reliable in vitro studies supported this conclusion. However, a chromosome aberration study in CHO cells did show positive effects without metabolic activation. Overall, sodium nitrate is considered negative in vitro.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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

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

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The reliability and summary were made based on the HPV program adopted during OECD SIAM 25 (October 2007). Only limited information is available on methods and results; the article is a review article of more than 200 investigated substances. The strains used are no standard strains as recommended by the OECD, not all necessary substitutions are added. Details on positive controls were not given

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

- Deficiency/Proficiency: histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: S. typhimurium TA92 and TA94 were also used.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix (polychlorinated biphenyls induced, KC-400)

Test concentrations with justification for top dose:

5 mg/plate (highest dose tested), 6 different concentrations (not given)

Vehicle / solvent:

phosphate buffer

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days

NUMBER OF REPLICATIONS:2

Evaluation criteria:

The result was considered positive if the number of revertant colonies found was twice or more that of the control. If no dose response was detected, additional experiments using different doses were performed.

Statistics:

not indicated

Key result

Species / strain:

S. typhimurium, other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Sodium nitrate is not mutagenic in the Ames test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The reliability and summary were made based on the HPV program adopted during OECD SIAM 25 (October 2007). Only limited information is available on methods and results; the article is a review article of more than 200 investigated substances. The strains used are no standard strains as recommended by the OECD, not all necessary substitutions are added. Details on positive controls were not given. No GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

no metabolic activation used,
cytotoxicity not measured,
exposure 24 and 48 h
100 metaphases investigated

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster fibroblast cell line, CHL

Metabolic activation:

without

Test concentrations with justification for top dose:

5 mg/ml (highest dose tested),
maximum dose selected in a preliminary test (not reported)
3 different concentrations (not given)

Vehicle / solvent:

physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

- Exposure duration: 24 and 48 hr
- Colcemid was added 2 h prior to cell harvesting
- cells fized with acetic-acid methanol
- stained with Giemsa
- 100 metaphases observed

Evaluation criteria:

The result was considered to be negative if the incidence of aberrations was less than 4.9%, equivocal if it was between 5.0 and 9.9% and positive if it was more than 10%.

Statistics:

For a quantitative evaluation of the clastogenic potential, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed.
In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/ml).

Key result

Species / strain:

other: Chinese hamster fibroblast cell line, CHL

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

24% Struct. aberr at 48hr

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

other: incidence of aberrations usually less than 3.0%

Untreated negative controls validity:

other: incidence of aberrations usually less than 3.0%

Positive controls validity:

not specified

Additional information on results:

D20 5.73 mg/ml
TR 0.8
Also positive at 4.0 mg/ml at both 24 hr (10.0%) and 48 hour (10.0%).

Conclusions:

may cause structural chromosomal aberrations, when incubated for 24 and 48 hours.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo chromosomal aberration and micronucleus test, UDS test, sperm abnormalitiy test, heritable translocation test all showed no genotoxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

heritable translocation test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The reliability and summary were made based on the HPV program adopted during OECD SIAM 25 (October 2007). non GLP

Qualifier:

no guideline followed

Principles of method if other than guideline:

OECD 485 is not followed, not GLP, no positive control, less animals used

GLP compliance:

not specified

Type of assay:

heritable translocation assay

Species:

mouse

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-12 weeks
- Housing: in wire-topped polycarbonate cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

distilled water

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Post exposure period:

At 10 days after the last treatment, each male was mated with 2 virgin females. Mating interval was 7 days.
At 5 weeks after treatment, treated and control males were killed by cervical dislocation for cytogenetic analysis.

Remarks:

Doses / Concentrations:
600 and 1,200 mg NaNO3/kg/day
Basis:

No. of animals per sex per dose:

25 males

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

P1 generation:
- cytogenetic analysis performed
- spermatocytes collected from all males, analysed in diakinesis-metaphase I.
- 100 cells per male analysed

F1 generation:
- F1 males were killed at 10-12 weeks.
- cytogenetic analysis performed on up to 3 males per litter from treated P males, and in 25 F1 males from controls.
- spermatocytes collected, analysed in diakinesis-metaphase I.
- 50 cells per F1 male analysed

Details of tissue and slide preparation:

chromosomal preparations made by Evans' air drying method, stained by orcein.

Statistics:

Student's t-test

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

no translocations in P1 males

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

no heritable chromosomal translocations in F1 males

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Sex chromosomal univalency was significantly higher at both doses.

Conclusions:

The substance was not mutagenic: negative no translocations in P1 males, no heritable chromosomal translocations in F1 males

Sex chromosomal univalency was significantly higher at both doses.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo chromosomal aberration and micronucleus test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

non-GLP

Qualifier:

no guideline followed

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

other: mouse (micronucleus evaluation) and rat (chromosomal aberration)

Strain:

other: Swiss mice, Wistar rats

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Biobreeding institute of Hygiene, Jassy.
- Age at study initiation: 10-14 weeks
- Diet: milk, bread, oats, carrot, sun flower seeds

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19
- Humidity (%): 40-50

Route of administration:

oral: gavage

Vehicle:

distilled water

Duration of treatment / exposure:

Twice in 24 hours (8 mice/group for micronuclei evaluation, animals killed 6 hr after last dose)
Twice in 24 hours (4 rats/group for chromosomal aberration analysis, animals killed 24 hr after last dose)
Daily for 2 weeks (6 rats/group for subacute test for chromosomal aberration analysis, animals killed 24 hr after last dose)

Remarks:

Doses / Concentrations:
108, 323, 969 and 2,906 mg/kg bw (sodium nitrate) 78.5, 235.5, 706.6 and 2120 mg/kg bw (nitrate)
Basis:

No. of animals per sex per dose:

8 mice/group for micronuclei evaluation, animals killed 24 hr after last dose
4 rats/group for chromosomal aberration analysis, animals killed 24 hr after last dose
6 rats/group for subacute test for chromosomal aberration analysis, animals killed 24 hr after last dose

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

micronuclei evaluation (mice) and chromosomal aberration analysis (rats)

Details of tissue and slide preparation:

The bone marrow preparations for chromosomal aberrations analysis were made according to Killian et al 1977. 50 metaphase cells per animal.
The preparations for MN analysis by the method of Schmid 1975. PCEs and NCEs were counted, at least 1000 polychromatic cells per mouse.
Slides were scored blindly.

Statistics:

chi-sqaure analysis, compared to control group.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Remarks:

indication for a cytotoxic effect of the nitrate in the marrow.

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

After acute exposure to nitrate, no significant difference between the frequency of chromosomal aberrations in the exposed and control groups was detected in either rats or mice with the exception of mice receiving a dose of 706.6 mg NO₃/kg. Rats treated for two weeks and long sampling time (24 hr) had significant increases in aberrant metaphases. Due to these shortcomings, 2 weeks of treatment and too long sampling, the positive efects may have been induced by this and can be disregarded. After acute exposure of mice, the frequency of micronucleated polychromatic erythrocytes increased significantly only at the lower doses of nitrate. This occurrence was probably masked at the higher doses by the invasion of the marrow by peripheral blood, which probably indicates a cytotoxic effect of the nitrate in the marrow. The sampling time was only 6 hr, too short, even though positive results were observed.

Positive controls were not included as required under OECD test guidelines; repeated dosing for 2 weeks is inconsistent with OECD TG 475.

Reference 3

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Sperm Abnormality

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The reliability and summary were made based on the HPV program adopted during OECD SIAM 25 (October 2007).The study was not performed according to an OECD guideline. Only 1 exposure time (3 days) was used. However, negative and positive controls are reported and the results clearly indicate that treatment of mice with sodium nitrate did not result in sperm-head abnormality.

Qualifier:

no guideline followed

Principles of method if other than guideline:

No guideline indicated

GLP compliance:

not specified

Species:

mouse

Strain:

C3H

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

11 and 17 days after treatment mice were killed.

Frequency of treatment:

3 times

Post exposure period:

Not indicated.

Remarks:

Doses / Concentrations:
600 and 1200 mg/kg/day
Basis:

No. of animals per sex per dose:

5 males/dose group

Control animals:

other: Vehicle control: water (same treatment as test substance treated groups). Positive control: MMS 75 mg/kg/day (i.p.) for 5 days.

Tissues and cell types examined:

caudal sperm

Key result

Sex:

male

Genotoxicity:

not determined

Remarks:

Sperm abnormality was determined

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

EFFECTS:

 

Dose (mg/kg/day)	Number of abnormal sperm heads
	11 days after treatment	17 days after treatment
control	16.0 ± 5.2	16.2 ± 3.2
600	14.8 ± 3.1	17.8 ± 5.5
1200	22.4 ± 8.7	25.8 ± 10.8
MMS (75)	33.2 ± 6.1**	47.0 ± 43.5

Sodium nitrate did not induce abnormalities in the sperm head of treated mice. No effects on rate of spermatogenesis.

STATISTICAL RESULTS:

No significant increases.

Conclusions:

No sperm abnormality was been observed.

Reference 4

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

No guideline indicated. The study was not performed according to the OECD guideline. Only 1 exposure time (3 days) was used, no clinical observations were made, and no reason was given for 1200 mg/kg/day as highest dose.

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Species:

mouse

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-12 weeks
- Housing: in wire-topped polycarbonate cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

distilled water

Details on exposure:

dose levels sodium nitrate: 600 and 1200 mg/kg/day
dosed with sodium nitrate or vehicle control 3 times: 48, 24 and 1.5 h prior to thymidine injection
3H deoxy-thymidine injected into testis
positive control MMS injected immediately after thymidine injection

Duration of treatment / exposure:

17 days after treatment mice were killed.

Frequency of treatment:

3 times, 48, 24 and 1.5 h prior to thymidine injection.

Remarks:

Doses / Concentrations:
600 and 1200 mg/kg/day
Basis:

No. of animals per sex per dose:

5 males/dose group.

Control animals:

yes, concurrent vehicle

other: Positive control: methyl methanesulfonate, MMS 75 mg/kg (i.p.).

Positive control(s):

methyl methanesulfonate, MMS 75 mg/kg (i.p.).

Tissues and cell types examined:

Unscheduled DNA synthesis was determined in early to mid spermatids from epididymis.
Organs examined at necropsy: caudal sperm

Evaluation criteria:

The UDS levels were presented as the incorporation of3H-deoxythymidine ([3H}dThd, in dpm) per 10 million sperms.

Statistics:

Student's t-test

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

GENOTOXIC EFFECTS:

 

Dose (mg/kg/day)	Incorporation of [^3H]dThd (in dpm/10^6sperm) in male mouse germ cells determined 17 days after treatment
control	9.2 ± 2.1
600	8.8 ± 2.3
1200	9.0 ± 1.2
MMS (75)	914.4 ± 168.7

No effects on rate of spermatogenesis.

 

STATISTICAL RESULTS:

No significant increases.

Conclusions:

no unscheduled DNA synthesis was demonstrated

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

Sodium nitrate is not mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, TA 92 and TA 94 with and without metabolic activation. No chromosomal aberrations or micronuclei were induced (not indicated whether metabolic activation was used). However, a chromosome aberration study in CHO cells did show positive effects without metabolic activation. Therefore, a new study was performed, and in this reliable OECD 473 guideline chromosome aberration study sodium nitrate did not show genotoxicity in human lymphocytes with or without metabolic activation. Several less reliable in vitro studies supported this conclusion.

In vivo studies

An in vivo chromosomal aberration and micronucleus test was negative. No unscheduled DNA synthesis was demonstrated in an UDS test. No sperm abnormalities or effect on rate of spermatogenesis were induced. Sodium nitrate was not mutagenic in a heritable translocation test. In a chromosome aberration/micronucleus study in rats and mice, no increase in chromosome aberrations were found when animals were treated twice. However, an increase in aberrant metaphases was observed in rats that were treated for 2 weeks with a 24 hour sampling. Such a repeated dosing and long sampling time is not according requirements, and as studies with acute dosing do not show positive findings with regard to chromosome aberrations this positive finding is considered not reliable. However, in the micronucleus study with the mice with a short sampling time (6 hours), two acute doses resulted already in an induction of micronuclei.

Based on all genotoxicity studies present, together with the findings that sodium nitrate does not induce tumor formation at high doses (see carcinogenicity section), it is concluded that sodium nitrate is not genotoxic.

Justification for selection of genetic toxicity endpoint

Several in vitro and in vivo studies on the substance are avaialble.

Justification for classification or non-classification

According to Annex I of Regulation (EC) No. 1272/2008 sodium nitrate is not classified based on the available data showing negative results from in vitro mammalian mutagenicity assays which do not support the outcome of one positive in vivo heritable germ cell mutagenicity test.

.