Calcium nitrite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an OECD Test Guideline 471 study, to GLP, the test material (DCI; 34% calcium nitrite in water) showed evidence of mutagenic activity in Salmonella typhimurium strains TA100 and TA1535 (but not in TA98 or TA1537) in the presence and absence of mammalian metabolic activation (Kitching, 1997).

 

No mammalian cell in vitro genotoxicity data with calcium nitrite were identified.

 

In early published (non-guideline) studies, with limited reporting, sodium nitrite induced mutagenic activity in cultured mouse cells (Kodama et al., 1976) and chromosome aberrations in Chinese hamster lung cells (Ishidate and Odashima, 1977), when tested in the absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4-16 January 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Good-quality study, conducted to GLP. The test material was a 34% solution of hydrated calcium nitrite. The deviation from the current relevant OECD guideline (e.g. inclusion also of TA102 or E.coli) would not impact the overall conclusion.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983 version

Deviations:

yes

Remarks:

Current Guideline recommends the use of at least 5 tester strains; the study used only 4, omitting a strain for detecting cross-linking mutagens (e.g. TA102 or E. coli WP2 uvrA). Calcium nitrite was only tested at up to 1700 µg/plate (cf. 5000 µg/plate).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29/12/1992 version

Deviations:

yes

Remarks:

Current Guideline recommends the use of at least 5 tester strains; the study used only 4, omitting a strain for detecting cross-linking mutagens (e.g. TA102 or E. coli WP2 uvrA). Calcium nitrite was only tested at up to 1700 µg/plate (cf. 5000 µg/plate).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1984 version

Deviations:

yes

Remarks:

Current Guideline recommends the use of at least 5 tester strains; the study used only 4, omitting a strain for detecting cross-linking mutagens (e.g. TA102 or E. coli WP2 uvrA). Calcium nitrite was only tested at up to 1700 µg/plate (cf. 5000 µg/plate).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 fraction obtained from the livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

50, 150, 500, 1500 or 5000 µg DCI/plate [top dose level is equivalent to about 1700 µg calcium nitrite/plate]

In a preliminary toxicity test, concentrations of 5, 50, 500 or 5000 µg/plate were tested in strains TA1535, TA1537, TA98 and TA100, with and without metabolic activation. Both the mutagenic and cytotoxic effects of the test material on these strains were analysed. As only slight cytotoxicity was observed at the highest concentration, subsequent mutagenicity testing on the four bacterial strains also used 5000 µg/plate as the top concentration; other concentrations used were 50, 150, 500 and 1500 µg/plate (with and without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test substance was supplied as an aqueous solution.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 days
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): no data

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: measured by the presence of an incomplete background lawn.

Evaluation criteria:

A test substance is considered positive (mutagenic) in the test if it induced at least a 2-fold increase (with some evidence of a positive dose-response) in the number of revertants with respect to the number induced by the solvent control in two separate experiments, with any of the tester strains, either with or without metabolic activation.

A test substance is considered to be negative (not mutagenic) if the total number of revertants in any tested strain at any concentration is not greater than 1.5 times the solvent control value, with or without metabolic activation.

Statistics:

No statistical evaluation was performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Large, dose-related increases in the revertant colony numbers were observed in both mutation experiments

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Large, dose-related increases in the revertant colony numbers were observed in both mutation experiments

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: The test material caused slight cytotoxicity (incomplete bacterial lawn) at the highest tested concentration (5000 µg DCI/plate) in all four tester strains, both with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: no data

Remarks on result:

other: Although the test material was applied at up to the recommended limit concentration of 5000 µg/plate, the highest actual tested concentration of calcium nitrite was 1700 µg/plate.

Conclusions:

In an OECD Test Guideline 471 study, to GLP, calcium nitrite (hydrate) showed evidence of mutagenic activity in Salmonella typhimurium strains TA100 and TA1535 (but not in TA98 or TA1537) in the presence and absence of mammalian metabolic activation.

Executive summary:

The genotoxic potential of calcium nitrite (hydrate) was assessed in an in vitro bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP.

 

In a preliminary toxicity test, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to test material concentrations of 0, 5, 50, 500 or 5000 µg/plate (equivalent to up to 1700 µg calcium nitrite/plate), both in the presence and absence of metabolic activation by rat liver fraction S9 (alongside appropriate vehicle controls). Only slight cytotoxicity was observed at the highest concentration only, therefore this was used as the highest test concentration in the mutagenicity assay.

Triplicate cultures of the four bacterial strains were exposed to the test material at concentrations of 50, 150, 500, 1500 or 5000 µg/plate, both with and without S9 (alongside appropriate vehicle and positive controls). These cultures were incubated for 72 hours at 37°C before being inspected for signs of cytotoxicity and for the incidence of revertant colonies. The main mutagenicity experiment was repeated using the same test material concentrations and following an identical procedure.

 

No signs of cytotoxicity were observed for any of the tested strains, at any of the tested concentrations, in either of the mutagenicity experiments. Large, dose-related increases in the number of revertant colonies were seen for S. typhimurium strains TA100 and TA1535 (with and without S9) in both mutagenicity experiments. No significant, dose-related increase was seen in S. typhimurium strains TA98 or TA1537 (both in the absence and presence of S9). The concurrent positive control substances demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

 

Under the conditions of this assay, calcium nitrite (hydrate) showed evidence of mutagenic activity in bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See 'Remarks'

Remarks:

Not to current international guidelines. Used a non-standard cell line; only tested without mammalian metabolic activation; no information provided regarding cytotoxicity, choice of concentrations, replicates and statistical evaluation. Nevertheless, the positive result from this study is useful in the overall weight of evidence.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Determination of mutagenicity in cultured mouse cells
- Short description of test conditions: Cells were treated with various concentrations of sodium nitrite (in minimal essential medium (MEM)) then plated with a selection agent before colony counting to determine mutation frequency.
- Parameters analysed / observed: Mutation frequency per 10^5 surviving cells

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

No data

Species / strain / cell type:

other: FM3A cells

Details on mammalian cell type (if applicable):

C3H mouse mammary carcinoma cell line.
FM3A cells were chosen because of their increased susceptibility to 8-AG-resistant mutation by the treatment of various mutagenic compounds.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Metabolic activation system:

Not applicable

Test concentrations with justification for top dose:

0.001, 0.0032 and 0.01 M (69, 218 and 690 μg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: minimal essential medium (MEM)
- Justification for choice of solvent/vehicle:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 2 days
- Expression time (cells in growth medium): inoculated directly on the 0.5% agarose medium (with or without 20 μg/mL 8-AG)
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): 8-azaguanine (8-AG)

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: no data

- OTHER: FM3A cells were grown in suspension in Eagle's MEM, containing 10% fetal bovine serum and antibiotics. Sodium nitrite was dissolved in MEM and diluted in the medium and sterilized through membrane filter before use.

Rationale for test conditions:

No further details

Evaluation criteria:

No further details

Statistics:

No further details

Species / strain:

other: FM3A cells (C3H mouse mammary carcinoma cell line)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

The mutation frequency per 10^5 surviving cells was 1.2 in controls, 4.7 at 69 μg/mL, 7.4 at 218 μg/mL and 59.2 at 690 μg/mL. Consequently, the ratio of the treated/control mutation frequencies was 3.9 at 69 μg/mL, 6.2 at 218 μg/mL and 49.3 at 690 μg/mL. These results indicate a dose-dependent increase of the mutation frequency.

Conclusions:

In an early published (non-guideline) study, with limited reporting, sodium nitrite was mutagenic to cultured mouse cells when tested in the absence of metabolic activation.

Executive summary:

In a non-guideline mammalian cell gene mutation assay involving cultured FM3A cells (a C3H mouse mammary cell line), sodium nitrite was tested in the absence of a metabolic activation system. Cells were treated with concentrations of 0.001, 0.0032 or 0.01 M (69, 218 or 690 μg/mL) sodium nitrite (in minimal essential medium) for 2 days before plating with the selection agent (8-azaguanine) for 12 days. Colonies were counted to determine the mutation frequency per 10^5 surviving cells.

 

The mutation frequency per 10^5 surviving cells was 1.2 in controls, 4.7 at 69 μg/mL, 7.4 at 218 μg/mL and 59.2 at 690 μg/mL. Consequently, the ratio of the treated/control mutation frequencies was 3.9 at 69 μg/mL, 6.2 at 218 μg/mL and 49.3 at 690 μg/mL. These results indicate a dose-dependent increase of the mutation frequency.

 

In conclusion, the test substance induced gene mutations in the FM3A cell line when tested in the absence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See 'Remarks'

Remarks:

Not to current international guidelines. Only tested without mammalian metabolic activation. Limited reporting, including a lack of information/clarity provided regarding cytotoxicity, choice of concentrations, replicates and statistical evaluation. Nevertheless, the positive result from this study is useful in the overall weight of evidence.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Determination of cytogenicity (chromosome aberrations) in Chinese hamster lung (CHL) cells
- Short description of test conditions: Following a growth inhibition test, three concentrations of the test material were added to 3-day old CHL cultures in the absence of S9. Chromosome aberrations and the types of breaks were subsequently recorded.
- Parameters analysed / observed:

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

other: Chinese hamster lung fibroblast cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: A clonal sub-line of a Chinese hamster fibroblast cell line (CHL) was used. This cell line was originally established from the lung of a young adult by Dr. T. Utakoji, Cancer Institute, Tokyo.
- Suitability of cells: no data
- Cell cycle length, doubling time or proliferation index: doubling time of 18.2 hours at their exponential growth at 37 deg C in a 5% CO2 atmosphere
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: 5-day passages
- Methods for maintenance in cell culture if applicable: no data
- Modal number of chromosomes: 25
- Normal (negative control) cell cycle time: no data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's minimal essential medium (MEM) supplemented with 10% calf serum
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically 'cleansed' against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Metabolic activation system:

Not applicable

Test concentrations with justification for top dose:

A total of three concentrations, including the "50% inhibition dose" (0.53 mg/mL; estimated in the growth inhibition test), were evidently tested. The additional concentrations tested are not clear from this early publication, but may have been 0.25 and 1.0 mg/mL [as cited in OECD, 2005].

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 24 hours (continuous treatment to sampling)
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: no data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were treated with colcemid (0.2 μg/mL) for 2 hours and, after trypsinisation, were incubated in 0.075 M KCl hypotonic solution for 15 min at 37 oC. Cells were fixed with ice-cold fixative (methanol/glacial acetic acid 3:1 v/v) which was changed three times. A few drops of the suspension were placed on clean dry slides, which were stained with 1% Giemsa's buffered solution for 20 min.

NUMBER OF CELLS EVALUATED: no data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: 50% growth inhibition estimated, following several doses added to 3-day old cultures. Colour absorption values were used to calculate relative cell densities.

OTHER EXAMINATIONS:
- Determination of polyploidy: only for saline and untreated controls
- Determination of endoreplication: no data

- OTHER: Types of aberration were classified into 5 groups: chromatid gaps, chromatid breaks, chromatid/chromosome translocation, ring formation and fragmentation/puverisation. Breaks less than the width of a sister chromatid were designated as gaps.

Evaluation criteria:

CHL cells commonly have less than 3.0% cells with chromosome aberrations. The test item was determined to be negative (-) if less than 4.9% of the aberration was detected even when doses of the agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious (±) if 5.0-9.9%, and positive if 10.0-19.9% (+), 20.0-49.9% (++) or more than 50.0% (+++). When no reasonable dose response was obtained, additional experiments with different doses were carried out to confirm its reproducibility.

Key result

Species / strain:

other: Chinese hamster lung fibroblast cell line

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

The 50% inhibition dose was evidently 0.53 mg/mL, associated with 21.0% chromosome aberrations at 24 hours. Among the changes observed were: chromatid gaps, chromatid/chromosome breaks and translocations.

Conclusions:

In an early published (non-guideline) study, with limited reporting, sodium nitrite induced chromosome aberrations in Chinese hamster lung cells in the absence of metabolic activation.

Executive summary:

The clastogenicity of sodium nitrite was assessed in a non-guideline in vitro study using Chinese hamster lung (CHL) fibroblasts. Cells were treated with the test substance (dissolved in saline) for 24 hours at 0.53 mg/mL (the 50% inhibition dose) in the absence of a metabolic activation system (alongside appropriate vehicle and untreated controls). Two additional concentrations were tested [these may have been 0.25 and 1.0 mg/mL, according to the OECD (2005)]. Fixation and Giemsa staining preceded the recording of the number of cells with chromosome aberrations.

Following sodium nitrite treatment, evidently 21.0% of CHL cells had chromosome aberrations [it is unclear whether this figure relates to the 50% inhibition concentration or to an average of all three concentrations]. Sodium nitrite was determined to be clastogenic in CHL cells in the absence of S9.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data with calcium nitrite were identified.

 

A mammalian erythrocyte micronucleus test was conducted (according to the NTP test protocol) using animals from a 14-week GLP repeated dose oral toxicity study with sodium nitrite in mice. No significant increase in the frequency of micronuclei in peripheral blood were reported following administration of sodium nitrite via drinking water at concentrations of up to 5000 ppm (respective dose levels of 990 and 1230 mg/kg bw/day for males and females) (NTP, 2001).

 

In two mammalian erythrocyte micronucleus tests, both conducted according to the NTP test protocol, sodium nitrite did not significantly increase the frequency of micronuclei in the bone marrow of male rats and mice, following intraperitoneal injection for 3 days at up to respective doses of 100 and 125 mg/kg bw/day (NTP, 2001).

 

Gavage treatment of male mice with sodium nitrite at up to 120 mg/kg bw/day for 14 days, prior to mating with untreated females, did not induce heritable translocations in male offspring (Alavantic et al., 1988).

 

In a mammalian comet assay, single gavage administration of sodium nitrite at 100 mg/kg bw to male mice did not significantly increase DNA damage in any of eight tested organs (Ohsawa et al., 2003).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Good-quality study conducted according to the NTP test protocol, and to GLP. Some limitations in reporting detail.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Mouse peripheral blood micronucleus NTP test protocol

Version / remarks:

As described by MacGregor et al. (1990).

MacGregor JT, Wehr CM, Henika PR and Shelby MD (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14, 513-522.

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 6 weeks
- Weight at study initiation: no data
- Assigned to test groups randomly: Animals were distributed randomly into groups of approximately equal initial mean body weights
- Fasting period before study: no data
- Housing: Solid-bottom polycarbonate cages (1 animal/cage), changed weekly; rotated every 2 weeks
- Diet (e.g. ad libitum): NIH-07 open formula powdered diet, available ad libitum, changed weekly
- Water (e.g. ad libitum): Charcoal-filtered deionized water, available ad libitum and changed twice weekly
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3°F (20.6-23.9°C)
- Humidity (%): 50 ± 15%
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 August 1989 To: 6 November 1989 (males) and 7 November 1989 (females)

Route of administration:

oral: drinking water

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 375, 750, 1500, 3000 or 5000 ppm
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 2 weeks by mixing sodium nitrite with water

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

Continuously

Post exposure period:

None (peripheral blood sampling/necropsy on day of last exposure)

Dose / conc.:

375 ppm

Remarks:

Equivalent to approximately 90 mg/kg bw/day (males) and 120 mg/kg bw/day (females).

Dose / conc.:

750 ppm

Remarks:

Equivalent to approximately 190 mg/kg bw/day (males) and 240 mg/kg bw/day (females).

Dose / conc.:

1 500 ppm

Remarks:

Equivalent to approximately 345 mg/kg bw/day (males) and 445 mg/kg bw/day (females).

Dose / conc.:

3 000 ppm

Remarks:

Equivalent to approximately 750 mg/kg bw/day (males) and 840 mg/kg bw/day (females).

Dose / conc.:

5 000 ppm

Remarks:

Equivalent to approximately 990 mg/kg bw/day (males) and 1230 mg/kg bw/day (females).

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Normochromatic erythrocytes (NCEs) from peripheral blood

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: mice from all treatment groups were scored for micronuclei

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): At the end of the 14-week treatment period, peripheral blood samples were obtained from 10 male and 10 female mice per group.

DETAILS OF SLIDE PREPARATION: Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group.

Evaluation criteria:

An experiment is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups (i.e. P ≤ 0.005 for males and females).

Statistics:

Mean numbers of micronucleated NCEs (per 1000 NCEs) were calculated along with standard errors. Significance of micronucleated NCEs/1000 NCEs tested by the one-tailed trend test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Extramedullary haematopoiesis observed in the spleen of males and females exposed at and above respective nominal concentrations of 3000 and 1500 ppm; males also displayed testes degeneration at the two highest concentrations

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

No significant (or dose-related) increase in the frequency of micronucleated NCEs was observed in the treated groups, compared to the control animals. As such, there was no indication of chromosome damage in this test.

Conclusions:

A mammalian erythrocyte micronucleus test was conducted (according to the NTP test protocol) using animals from a 14-week GLP repeated dose oral toxicity study on sodium nitrite in mice. No significant increase in the frequency of micronuclei in peripheral blood were reported following administration of sodium nitrite via drinking water at concentrations of up to 5000 ppm (respective dose levels of 990 and 1230 mg/kg bw/day for males and females).

Executive summary:

A mammalian erythrocyte micronucleus test was conducted according to the NTP test protocol using animals from a GLP subchronic oral toxicity study. In the main repeated dose study, sodium nitrite was administered daily via drinking water to mice (10/sex/group) at 375, 750, 1500, 3000 or 5000 ppm (equivalent to approximate dose levels of 90, 190, 345, 750 or 990 mg/kg bw/day for males and 120, 240, 445, 840 or 1230 mg/kg bw/day for females) for 14 weeks. Control animals received vehicle only. Peripheral blood samples were obtained at the end of the treatment period, fixed and stained prior to analysis. The frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) was calculated.

 

No significant increase in the frequency of micronucleated NCEs was observed in any of the treated dose groups. Extramedullary haematopoiesis was observed in the spleen of males and females exposed at and above respective nominal concentrations of 3000 and 1500 ppm, while males also displayed testes degeneration at the two highest concentrations [See Repeated dose toxicity section for details].

 

It is concluded that sodium nitrite failed to induce an increase in the frequency of micronuclei in the blood of mice following administration via drinking water at concentrations of up to 5000 ppm (about 990-1230 mg/kg bw/day in males and females respectively) for 14 weeks.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Good quality-study conducted according to the NTP test protocol. Some limitations in reporting detail.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: Rat and mouse bone marrow micronucleus NTP test protocol

Version / remarks:

The standard three-exposure protocol is described in detail by Shelby et al. (1993).

Shelby MD, Erexson GL, Hook GJ and Tice RR (1993). Evaluation of a three-exposure mouse bone marrow micronucleus protocol: Results with 49 chemicals. Environ. Mol. Mutagen. 21, 160-179.

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

other: F344/N

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: presumably Taconic Farms (Germantown, NY)
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): presumably ad libitum
- Water (e.g. ad libitum): presumably ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): presumably 72 ± 3°F (20.6-23.9°C)
- Humidity (%): presumably 50 ± 15%
- Air changes (per hr): presumably ≥10
- Photoperiod (hrs dark / hrs light): presumably 12/12

IN-LIFE DATES: No data

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: phosphate-buffered saline (PBS)
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data

Details on exposure:

The test material was dissolved in PBS and injected intraperitoneally.

Duration of treatment / exposure:

3 days (three injections at 24-hour intervals)

Frequency of treatment:

Daily

Post exposure period:

Animals were killed 24 hours after the third injection

Dose / conc.:

6.25 mg/kg bw/day (actual dose received)

Remarks:

Inital test only.

Dose / conc.:

12.5 mg/kg bw/day (actual dose received)

Remarks:

Inital test only.

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

tested in two separate experiments

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

tested in two separate experiments

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Inital test only.

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Inital test only. Lethal dose

No. of animals per sex per dose:

5 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal
- Doses / concentrations: 25 mg/kg bw

Tissues and cell types examined:

Bone marrow cells from femurs

Details of tissue and slide preparation:

The animals were killed 24 hours after the third injection and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained. 2000 polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in up to 5 animals/dose group.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups (i.e. P ≤ 0.005 for trial 1 and ≤ 0.012 for trial 2). A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

The frequency of micronucleated cells among PCEs was analysed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochrane-Armitage trend test followed by pairwise comparisons between each dosed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

All the animals died at 200 mg/kg bw/day in the initial test.

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

No significant increase in the frequency of micronucleated PCEs was observed in any of the dose groups. The initial trial was judged to be positive, based on the trend test (P=0.001; significant at P ≤ 0.025); however, a repeat trial involving doses of 0, 25 or 50 mg/kg bw was negative (P=0.500), and the rat bone marrow micronucleus test with sodium nitrite was judged to be negative overall.

Conclusions:

In a mammalian erythrocyte micronucleus test, conducted according to the NTP test protocol, sodium nitrite did not significantly increase the frequency of micronuclei in the bone marrow of male rats, following intraperitoneal injection for 3 days.

Executive summary:

A mammalian erythrocyte micronucleus test was conducted according to the NTP test protocol. Sodium nitrite (in phosphate-buffered saline) was administered to male rats (5/group) by intraperitoneal injection at 6.25-200 mg/kg bw/day for 3 days. Positive control animals received cyclophosphamide similarly at 25 mg/kg bw/day, while negative control animals received vehicle only. In a repeat experiment, sodium nitrite doses of 25 and 50 mg/kg bw/day were used, along with the associated control substances. Animals were euthanised 24 hours after the last injection and blood smears were prepared, fixed and stained. Two thousand polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

 

No significant increase in the frequency of micronucleated PCEs was observed in any of the dose groups. The initial experiment was judged to be positive, based on the trend test (P=0.001; significant at P ≤ 0.025); however, the repeat experiment was negative (P=0.500), and the rat bone marrow micronucleus test with sodium nitrite was judged to be negative overall. The high dose animals died in the initial test.

In conclusion, in a NTP study, sodium nitrite did not produce an increase in micronuclei in the bone marrow of male rats following intraperitoneal injection for 3 consecutive days.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Good-quality study conducted according to the NTP test protocol. Some limitations in reporting detail.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: Rat and mouse bone marrow micronucleus NTP test protocol

Version / remarks:

The standard three-exposure protocol is described in detail by Shelby et al. (1993).

Shelby MD, Erexson GL, Hook GJ and Tice RR (1993). Evaluation of a three-exposure mouse bone marrow micronucleus protocol: Results with 49 chemicals. Environ. Mol. Mutagen. 21, 160-179.

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: presumably Taconic Farms (Germantown, NY)
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): presumably ad libitum
- Water (e.g. ad libitum): presumably ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): presumably 72 ± 3°F (20.6-23.9°C)
- Humidity (%): presumably 50 ± 15%
- Air changes (per hr): presumably ≥10
- Photoperiod (hrs dark / hrs light): presumably 12/12

IN-LIFE DATES: No data

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: phosphate-buffered saline (PBS)
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data

Details on exposure:

The test material was dissolved in PBS and injected intraperitoneally.

Duration of treatment / exposure:

3 days (three injections at 24-hour intervals)

Frequency of treatment:

Daily

Post exposure period:

Animals were killed 24 hours after the third injection

Dose / conc.:

7.81 mg/kg bw/day (actual dose received)

Dose / conc.:

15.63 mg/kg bw/day (actual dose received)

Dose / conc.:

31.25 mg/kg bw/day (actual dose received)

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Lethal dose

No. of animals per sex per dose:

5 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Bone marrow cells from femurs

Details of tissue and slide preparation:

The animals were killed 24 hours after the third injection and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained. 2000 polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in up to 5 animals/dose group.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups (i.e. P ≤ 0.005).

Statistics:

Mean numbers of micronucleated PCEs (per 1000 PCEs) were calculated along with standard errors. Significance of micronucleated PCEs/1000 PCEs tested by the one-tailed trend test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

All the animals died at 250 mg/kg bw/day.

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

No significant increase in the frequency of micronucleated PCEs was observed in any of the dose groups. The experiment was considered to be negative, based on the trend test (P=0.343; significant at P ≤0.025) and P values for individual groups which were in excess of 0.005.

Conclusions:

In a mammalian erythrocyte micronucleus test, conducted according to the NTP test protocol, sodium nitrite did not significantly increase the frequency of micronuclei in the bone marrow of male mice, following intraperitoneal injection for 3 days.

Executive summary:

A mammalian erythrocyte micronucleus test was conducted according to the NTP test protocol. Sodium nitrite (in phosphate-buffered saline) was administered to male mice (5/group) by intraperitoneal injection at 7.81-250 mg/kg bw/day for 3 days. Positive control animals received cyclophosphamide similarly at 50 mg/kg bw/day, while negative control animals received vehicle only. Animals were euthanised 24 hours after the last injection and blood smears were prepared, fixed and stained. Two thousand polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

 

No significant increase in the frequency of micronucleated PCEs was observed in any of the dose groups. The experiment was judged to be negative, based on the trend test (P=0.343; significant at P ≤ 0.025) and the P values for individual groups (all in excess of 0.005). The high dose animals died in the experiment.

In conclusion, in a NTP study, sodium nitrite did not produce an increase in micronuclei in the bone marrow of male mice following intraperitoneal injection for 3 consecutive days.

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Generally well-conducted study, albeit with only two dose levels. The OECD considered this study to be reliability 2 ("valid with restrictions").

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Determination of cytogenicity (heritable translocations) in the germ cells of male mice and their offspring
- Short description of test conditions: Male mice were gavaged with two concentrations of the test material daily for 2 weeks before mating with untreated females.
- Parameters analysed / observed: heritable translocations (F1 animals), cytogenetic analysis (P animals) and sperm abnormalities (P and F1 animals).

GLP compliance:

not specified

Type of assay:

heritable translocation assay

Species:

mouse

Strain:

other: hybrid strain C3H x 101

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10-12 weeks (males and females)
- Weight at study initiation: no data
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: "wire-topped" polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data

Details on exposure:

The test material was dissolved in distilled water and administered by stomach intubation.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Post exposure period:

Males were killed 5 weeks after treatment

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

This dose was determined as "moderate comparing allowed quantities according to the Book of Regulations for meat products quality".

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

This dose was determined as the "maximum dose tolerated or the highest dose at which all animals survived".

No. of animals per sex per dose:

25 males

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Spermatocytes and cells from the cauda epididymis

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: not applicable

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): F1 males were analysed at maturation (10-12 weeks)

DETAILS OF SLIDE PREPARATION:
Heritable translocation test: Chromosome preparations were made using Evans' air-drying method and stained by orcein; 50 cells/animal were examined. Each slide was also treated with the C-banding technique.
Cytogenetic analysis of P males: 100 cells/animal were examined, in the same way as for F1 males (hertiable translocation test).
Sperm abnormality test: Analysed cells from the cauda epididymis were generated from treated differentiating spermatogonia (mostly B-type); 100 and 500 cells/animal were analysed for P and F1 males, respectively.

METHOD OF ANALYSIS: no data

Evaluation criteria:

No data

Statistics:

Statistical processing of results was done using Student's t-test (by χ squared in one case).

Sex:

male

Genotoxicity:

negative

Remarks:

no induction of translocations in P males

Toxicity:

yes

Remarks:

significant increase in abnormal sperm-head frequency

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not examined

Sex:

male

Genotoxicity:

negative

Remarks:

no induction of heritable translocations in F1 males

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Sex-chromosomal univalency was significantly increased at the higher dose level in P males, but was unaffected in F1 males.

A significant increase in abnormal sperm-head frequency was observed in parental males, though not in F1 males.

Morphological changes, including eyes, coat colour, testes and body weight, were unaffected in P and F1 males.

The high dose of sodium nitrite significantly reduced the number of fertile females while the low dose reduced both fertility and litter size (not statistically significant). In both treatment groups, the sex ratio in offspring from semisterile males (in matings where either one female was sterile or both females gave a significantly lower number of offspring relative to the average) deviated unusually towards an increased relative number of female offpsring.

The investigators considered that the lack of heritable translocations and heritable sperm abnormality, as well as the results of other work by the same investigtors on unscheduled DNA synthesis in spermatids of treated males, indicate that sodium nitrite was not mutagenic in the tested stages of germ cells of mice.

Conclusions:

Gavage treatment of male mice with sodium nitrite at up to 120 mg/kg bw/day for 14 days, prior to mating with untreated females, did not induce heritable translocations in male offspring.

Executive summary:

In a reliable (non-guideline) study, sodium nitrite was administered to male mice (25/group) by oral gavage at 0, 60 or 120 mg/kg bw/day for 14 days prior to mating with untreated females. Parental males were euthanised 5 weeks after treatment, while offspring were analysed at maturation (10-12 weeks). Parameters evaluated included heritable translocations (F1 animals), cytogenetic analysis (P animals) and sperm abnormalities (P and F1 animals).

There was no induction of heritable translocations in F1 males. Similarly, no translocations were apparent in parental males. A significant increase in abnormal sperm-head frequency was observed in parental males, though not in F1 males. In conclusion, sodium nitrite did not induce heritable translocations in the offspring of male mice following gavage treatment for 14 days.

Reference 5

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Generally well-conducted study, albeit with only a single relatively low dose level. Although somewhat limited, this study was used as part of EFSA's weight of evidence assessment and supports the findings of the well-conducted NTP investigations.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Determination of DNA damage using the comet assay in a variety of tissues of male mice following exposure to sodium nitrite
- Short description of test conditions: Male mice received a single gavage dose of sodium nitrite at 100 mg/kg bw before tissue sampling (and comet assay measurements) at 3 and 24 hours. [The relevant OECD guideline (TG 489) recommends the use of at least two dose levels in addition to the maximum (limit) dose (2000 mg/kg bw/day for administration periods of less than 14 days).]
- Parameters analysed / observed: DNA migration

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

other: ddY

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan SLC Co. (Shizuoka, Japan)
- Age at study initiation: 7 weeks
- Weight at study initiation: no data
- Assigned to test groups randomly: yes
- Fasting period before study: overnight
- Housing: no data
- Diet (e.g. ad libitum): commercial pellets MF ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 55-65
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data

Details on exposure:

The test material was dissolved in saline and administered by gastric intubation.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Not applicable

Post exposure period:

3 or 24 hours

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 males/group

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: not applicable

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were killed 3 or 24 hours after treatment and the relvant organs were excised for analysis.

DETAILS OF SLIDE PREPARATION: Slides were prepared from nuclei isolated by homogenisation and were placed in a chilled lysing solution for >60 minutes followed by a chilled alkaline solution for 10 minues (both solutions were kept at 0 deg C in the dark). Electrophoresis was conducted in the dark at 0 deg C for 15 minutes at 25 V and around 250 mA. Slides were neutralised and stained with ethidium bromide.

METHOD OF ANALYSIS: Fluorescence microscope

OTHER: 50 nuclei/slide were examined and comet length and head diameter were measured for 50 nuclei/organ/animal. Mean migration (difference between length and diameter) of 50 nuclei from each organ were calculated for each individual animal.

Evaluation criteria:

Upon statistical comparison of averages of DNA migration between treated and control animals, a P value less than 0.05 was considered statistically significant.

Statistics:

Dunnett test after one-way ANOVA. A P-value of less than 0.05 was considered statistically significant

Sex:

male

Genotoxicity:

negative

Remarks:

No statistically significant increase in DNA migration was reported for any of the organs tested at the 3 and 24 hour time points.

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: The EFSA Panel noted that no indication of genotoxicity of nitrite was observed in this study.

Additional information on results:

The EFSA Panel noted that the higher dose levels of sodium nitrite applied were lower than the maximum recommended.

Conclusions:

In a mammalian comet assay, single gavage administration of sodium nitrite at 100 mg/kg bw to male mice did not significantly increase DNA damage in any of eight tested organs.

Executive summary:

The genotoxic potential of sodium nitrite was investigated in a mammalian comet assay. After an overnight fast, male mice (4/group) received a single gavage dose of the test material (in saline) at 0 or 100 mg/kg bw (which is lower than the maximum recommended). Animals were euthanised 3 or 24 hours after treatment and samples from eight organs (stomach, colon, liver kidney, urinary bladder, lung, brain and bone marrow) were prepared for comet measurements following electrophoresis. Comet length and head diameter were measured for 50 nuclei/organ/animal and used to calculate mean DNA migration values.

No statistically significant increase in DNA migration was reported for any of the organs tested at the 3 and 24 hour time points. As such, the investigators concluded that sodium nitrite "did not yield DNA damage in any of the organs tested". The EFSA Panel noted that no indication of genotoxicity of nitrite was observed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable.

Additional information

The mutagenic potential of the test material (DCI; calcium nitrite (hydrate); 34% calcium nitrite in water) was assessed in an in vitro bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP. In a preliminary toxicity test, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to test material at concentrations of 0, 5, 50, 500 or 5000 µg/plate (equivalent to up to 1700 µg calcium nitrite/plate), both in the presence and absence of metabolic activation by rat liver S9 fraction, alongside appropriate vehicle controls. Slight cytotoxicity was observed at the highest concentration only, therefore 5000 µg/plate was used as the top test concentration in the mutagenicity tests. Triplicate cultures of the four bacterial strains were exposed to the test material at concentrations of 50, 150, 500, 1500 or 5000 µg/plate, both with and without S9, alongside appropriate vehicle and positive controls. These cultures were incubated for 72 hours at 37°C before being inspected for signs of cytotoxicity and for the incidence of revertant colonies. The main mutagenicity experiment was repeated using the same test material concentrations and following an identical procedure. No signs of cytotoxicity were observed for any of the tested strains, at any of the tested concentrations, in either of the mutagenicity experiments. Large, dose-related increases in the number of revertant colonies were seen for S. typhimurium strains TA100 and TA1535 (with and without S9) in both mutagenicity experiments. No significant, dose-related increase was seen in S. typhimurium strains TA98 or TA1537 (either in the absence or presence of S9). The concurrent positive control substances demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. Under the conditions of this assay, calcium nitrite (hydrate) showed evidence of mutagenic activity in bacteria with and without S9 (Kitching, 1997).

 

No other in vitro genotoxicity data with calcium nitrite were identified, notably no in vitro mammalian cell assays. As such, data on the closely-related sodium nitrite were considered suitable for use in a read-across approach. [See read-across justification report in IUCLID section 13 for details.]

 

Sodium nitrite was tested in a non-guideline mammalian cell gene mutation assay involving cultured FM3A cells (a C3H mouse mammary cell line), presumably in the absence of an exogenous metabolic activation system. Cells were treated with concentrations of 0.001, 0.0032 or 0.01 M (69, 218 or 690 μg/mL) sodium nitrite (in minimal essential medium) for 2 days before plating with the selection agent (8-azaguanine) for 12 days. Colonies were counted to determine the mutation frequency per 105 surviving cells. The mutation frequency per 10^5 surviving cells was 1.2 in controls, 4.7 at 69 μg/mL, 7.4 at 218 μg/mL and 59.2 at 690 μg/mL. Consequently, the ratio of the treated/control mutation frequencies was 3.9 at 69 μg/mL, 6.2 at 218 μg/mL and 49.3 at 690 μg/mL. These results indicate a concentration-dependent increase of the mutation frequency. No statistical analysis of the data was reported. In conclusion, sodium nitrite induced gene mutations in the FM3A cell line when tested in the apparent absence of added metabolic activation (Kodama et al., 1976).

 

The clastogenicity of sodium nitrite was assessed in an inadequately-reported non-guideline in vitro study using Chinese hamster lung (CHL) fibroblasts. Cells were treated with the test substance (dissolved in saline) for 24 hours at 0.53 mg/mL (the 50% inhibition dose), estimated during a preliminary investigation), presumably in the absence of an added metabolic activation fraction, alongside appropriate vehicle and untreated controls. Two additional concentrations were tested [these may have been 0.25 and 1.0 mg/mL, according to the 2005 OECD review]. Fixation and Giemsa staining preceded the recording of the number of cells with chromosome aberrations. Following sodium nitrite treatment, evidently 21.0% of CHL cells had chromosome aberrations (the incidence in controls was ≤ 1.1%) [it is unclear whether this figure relates to the 50% inhibition concentration or to an average of all three concentrations]. The observed changes included chromatid gaps, chromatid/chromosome breaks and translocations. Sodium nitrite was determined to be clastogenic in CHL cells in the absence of metabolic activation (Ishidate and Odashima, 1977).

 

A number of other (early) in vitro studies with sodium nitrite are available, and these have been summarised by various expert groups, including EFSA (2017), Health Canada (2013), IARC (2010) and the OECD (2005). These studies (predominantly >25 years old) show clear evidence of sodium nitrite’s in vitro mutagenic activity (both in bacteria and mammalian cells) and of its in vitro clastogenic activity (chromosome aberrations) in cultured mammalian cells, supporting the positive findings of the three in vitro studies summarised above.

 

No in vivo genotoxicity data with calcium nitrite are available. As such, certain in vivo genotoxicity studies on sodium nitrite, including three good-quality studies conducted under the auspices of the US National Toxicology Programme (NTP) (each assessing micronuclei induction), in addition to a germ cell translocation assay (assessing germ cell defects) and a comet assay (assessing DNA damage), were considered suitable for use in a read-across approach [see read-across justification report in IUCLID section 13 for details] and are described below.

 

In the first, a mammalian erythrocyte micronucleus test was conducted according to the NTP test protocol and to GLP, using animals from a subchronic NTP oral toxicity study. In the main repeated-dose study, sodium nitrite was provided daily via drinking water to mice (10/sex/group) at 375, 750, 1500, 3000 or 5000 ppm (equivalent to approximate dose levels of 90, 190, 345, 750 or 990 mg/kg bw/day for males and 120, 240, 445, 840 or 1230 mg/kg bw/day for females) for 14 weeks. Control animals received vehicle only. [Current NTP guidelines recommend inclusion of a positive control group or reference to a historical control range for the testing facility, neither of which were reported by the investigators; as such, this represents a slight deficiency in the test method, though this is not considered to affect the validity of the overall conclusion]. Peripheral blood samples were obtained at the end of the treatment period, fixed and stained prior to analysis. The frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) was calculated. No significant increase in the frequency of micronucleated NCEs was observed in any of the treated dose groups. Blood nitrite determinations performed within the framework of the same study (2-year investigation in mice) indicated the systemic exposure of treated animals to sodium nitrite at and above 1500 ppm [see Repeated-dose toxicity section for details]. It is concluded that sodium nitrite failed to induce an increase in the frequency of micronuclei in the blood of mice following administration via drinking water at concentrations of up to 5000 ppm (about 990-1230 mg/kg bw/day) for 14 weeks (NTP, 2001).

 

Two mammalian bone marrow micronucleus tests were conducted according to the NTP test protocol. Sodium nitrite (in phosphate-buffered saline) was administered to males (5/group) by intraperitoneal injection at 6.25-200 mg/kg bw/day (in rats) and 7.81-250 mg/kg bw/day (in mice) for 3 days. Positive control animals received cyclophosphamide similarly at 25 or 50 mg/kg bw/day, while negative control animals received vehicle only. In both studies, lethality [not further specified] was observed at the top dose tested (200 and 250 mg/kg bw/day in rats and mice, respectively). In a repeat experiment in rats only, ‘non-lethal’ sodium nitrite doses of 25 and 50 mg/kg bw/day were used, along with the associated control substances. Animals were euthanised 24 hours after the last injection and blood smears were prepared from bone marrow cells, fixed and stained. Two thousand polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. No significant increase in the frequency of micronucleated PCEs was observed in any of the individual dose groups, in rats or mice, compared to controls. In rats, the initial experiment was judged to be positive, based on the trend test (P=0.001; significant at P ≤ 0.025); however, the repeat experiment was negative based on the trend test (P=0.500) and following pairwise comparison with controls for both dose groups. Overall, the NTP investigators judged the rat bone marrow micronucleus test with sodium nitrite as negative. In conclusion, in these NTP studies, sodium nitrite did not produce an increase in micronuclei in the bone marrow of male rats or male mice following intraperitoneal injection for 3 consecutive days (NTP, 2001). Significant toxicity (lethality) was clearly seen at the higher doses in these studies (in both rats and mice), demonstrating systemic exposure of the test material. However, no toxicokinetic assessment was conducted to demonstrate exposure/toxicity of the bone marrow cells following intraperitoneal injection. Current guidelines recommend an assessment of the NCE/PCE ratio as a marker of bone marrow toxicity (and thus exposure). Such an assessment was evidently not conducted by the investigators (as it was not required by the NTP test protocol at the time) and represents a deficiency in the test method. Effects on erythropoietic activity at and above 1500 ppm in the 14-week mouse drinking water study [see Repeated-dose toxicity section for details] indicate the potential for bone marrow effects following systemic exposure to sodium nitrite (NTP, 2001), albeit it via a different exposure route, dose and study duration. An EFSA (2017) assessment of these studies noted that “no measurement of the PCE/NCE ratio in bone marrow was performed, and thus it is not possible to assess whether treatment determined a significant toxicity, and hence exposure, of the erythropoietic tissue. However, the Panel also noted that blood nitrite determinations performed within the framework of the same study highlighted the systemic exposure of treated animals both after single administration (6.26 mg/kg, in rats) and exposure to nitrite via drinking water (from 1500 mg/L onwards, in mice). Overall, despite the intraperitoneal route is usually considered not relevant for the assessment of genotoxic hazard related to oral exposure, as by-pass of first-pass metabolism may result in an irrelevant high internal exposure, the Panel noted that this study could be considered as a worst case in a weight of evidence evaluation of the genotoxicity of nitrite”.

 

In a reliable (non-guideline) study, sodium nitrite was administered to male mice (25/group) by oral gavage at 0, 60 or 120 mg/kg bw/day for 14 days prior to mating with untreated females. Parental males were euthanised 5 weeks after treatment, while offspring were analysed at maturation (10-12 weeks). Parameters evaluated included heritable translocations (F1 animals), cytogenetic analysis (P animals) and sperm abnormalities (P and F1 animals). There was no induction of heritable translocations in F1 males. Similarly, no translocations were apparent in parental males. A significant increase in abnormal sperm-head frequency was observed in parental males, though not in F1 males. In conclusion, sodium nitrite did not induce heritable translocations in the offspring of male mice following gavage treatment for 14 days (Alavantic et al., 1988).

 

The genotoxic potential of sodium nitrite was investigated in a mammalian comet assay. After an overnight fast, male mice (4/group) received a single gavage dose of the test material (in saline) at 0 or 100 mg/kg bw (which is lower than the maximum recommended). Animals were euthanised 3 or 24 hours after treatment and samples from eight organs (stomach, colon, liver kidney, urinary bladder, lung, brain and bone marrow) were prepared for comet measurements following electrophoresis. Comet length and head diameter were measured for 50 nuclei/organ/animal and used to calculate mean DNA migration values. No statistically significant increase in DNA migration was reported for any of the organs tested at the 3 and 24-hour time points. As such, the investigators concluded that sodium nitrite "did not yield DNA damage in any of the organs tested" (Ohsawa et al., 2003). The EFSA Panel noted that no indication of genotoxicity of nitrite was observed in this study (EFSA, 2017).

 

A number of other (early) in vivo studies with sodium nitrite are available, and these have been summarised by various expert groups, including EFSA (2017), Health Canada (2013), IARC (2010) and the OECD (2005). These other studies (predominantly >30 years old) show a mixture of positive and negative findings for clastogenic activity (micronuclei, sister-chromatid exchange, and chromosome aberrations) in rodents.

 

In humans, high intake of nitrate (which can interconvert with nitrite in vivo) was associated with an increased frequency of hypoxanthine-guanine phosphoribosyl transferase gene mutations in peripheral blood lymphocytes; similarly, high nitrate intake caused an increase in the number of chromosome aberrations in lymphocytes of children (IARC, 2010).

 

IARC (2010) noted that sodium nitrite was positive “in several assays for chromosome aberrations and micronucleus formation, both in vitro and in vivo”. Similarly, both Health Canada (2013) and the OECD (2005) considered there to be evidence of in vivo genotoxic potential for (sodium) nitrite. However, in the most recent and comprehensive of the Expert Group reports noted above, the EFSA Panel (2017) stated that “in vivo negative results were obtained in well-performed micronucleus assays in mice and rats, with measurable systemic exposure, after acute and subchronic administration of sodium nitrite. Lack of genotoxicity is also indicated by more limited data at the site of contact (stomach): these include a negative comet assay by gavage, although at a dose lower than the maximum recommended, the absence of oxidative DNA damage in forestomach epithelium following dietary exposure to nitrite, and the lack of tumour-initiating activity in two-stage stomach cancer model”. Overall, the Panel concluded that there was no indication of an in vivo genotoxic potential for sodium nitrite, and considered it appropriate to establish an acceptable daily intake (ADI) of 0.1 mg/kg bw (0.07 mg nitrite ion/kg bw; on the basis of increased methaemoglobin levels in repeated-dose studies in rodents) (EFSA, 2017). The WHO evidently also had no significant concern over the in vivo genotoxicity of [sodium] nitrite in establishing a guideline value of 3 mg nitrite/L in drinking water (based on epidemiological data again relating to methaemoglobin levels) (WHO, 2011).

Further, in chronic toxicity studies conducted under the auspices of the NTP, exposure of rats to sodium nitrite at up to 3000 ppm (about 130-150 mg/kg bw/day) via drinking water for 2 years produced no evidence of carcinogenicity; sodium nitrite was also non-carcinogenic in male mice exposed similarly (up to about 220 mg/kg bw/day), while females displayed an increased incidence of squamous cell papilloma or carcinoma (combined) of the forestomach at the highest tested dose level (about 165 mg/kg bw/day) (though this effect was not statistically significant) [see Carcinogenicity section for further details] (NTP, 2001). An EFSA (2017) citation of these studies reported the highest tested dose level in each case as the study NOAEL, indicating a lack of concern regarding carcinogenicity.

 

Sodium nitrite was not considered a reproductive toxicant in another NTP study, conducted according to the continuous breeding protocol, involving exposure of mice to sodium nitrite at up to 0.24% in drinking water [around 412-473 mg/kg bw/day] for a total of 105 days [see Toxicity to reproduction section for further details] (NTP, 1990). An EFSA (2017) citation of this study noted a lack of fertility/reproductive and developmental effects.

 

The substance-specific and read-across studies summarised above demonstrate an in vitro mutagenic potential for calcium nitrite (in bacteria) and for sodium nitrite (in bacteria and in mammalian cells). Although the potential to induce chromosome aberrations was also indicated in vitro for sodium nitrite, the available good-quality in vivo micronuclei studies conducted on sodium nitrite by the NTP provide some degree of reassurance regarding cytogenicity (although there is uncertainty around whether the tested substance actually reached the target tissue in the bone marrow studies in sufficient quantity). The identified expert group reviews on sodium nitrite, generally indicate both mutagenic and clastogenic activity in vitro; a mixture of positive and negative in vivo results was identified. Such potential was acknowledged by IARC (2010), Health Canada (2013) and OECD (2005) experts, but EFSA (2017) opted to dismiss the in vivo genotoxicity potential of sodium nitrite. In addition, the available heritable translocation assay and negative NTP chronic and fertility studies (all on sodium nitrite) provide a certain amount of reassurance as regards carcinogenicity and effects on germ cells.

 

Overall, the EFSA (2017) evaluation provides the most recent and comprehensive overview of the genotoxicity potential of sodium nitrite. This authoritative opinion from a respected European expert group, concluding that there was no indication of an in vivo genotoxic potential for sodium nitrite, coupled with the negative results in the good-quality NTP carcinogenicity and reproductive toxicity studies as well as the negative in vivo comet and heritable translocation (assessing germ cell defects) assays on sodium nitrite, indicates that calcium nitrite does not pose an in vivo genotoxicity concern.

 

 

References (not included in IUCLID ESRs)

 

Health Canada (2013). Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Nitrate and Nitrite. Catalogue No H144-13/2-2013E-PDF.http://healthycanadians.gc.ca/publications/healthy-living-vie-saine/water-nitrate-nitrite-eau/alt/water-nitrate-nitrite-eau-eng.pdf?_ga=2.222634333.774105563.1517223052-337288668.1517223052

 

IARC (2010). International Agency for Research on Cancer. Ingested nitrate and nitrite and cyanobacterial peptide toxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Volume 94, 14-21 June 2006.http://monographs.iarc.fr/ENG/Monographs/vol94/mono94.pdf

 

WHO (2011). World Health Organization. Nitrate and nitrite in drinking water. Background document for development of WHO guidelines for drinking-water quality. WHO/SDE/WSH/07.01/16/Rev/1.http://www.who.int/water_sanitation_health/dwq/chemicals/nitratenitrite2ndadd.pdf

Justification for classification or non-classification

Based on the existing data set, with particular reference to the reassuring results in an in vivo heritable translocation assay in male mice (specifically a lack of heritable defects in the germ cells of offspring following repeated oral exposure to sodium nitrite), calcium nitrite does not currently meet the criteria for classification as a germ cell mutagen (category 1A, 1B or 2) under EU CLP criteria (EC 1272/2008). In support, an EFSA Panel concluded that the available information described above does not indicate an in vivo genotoxic potential for [the structurally related substances] sodium and potassium nitrite (EFSA, 2017).