Calcium nitrite

Genetic toxicity: in vivo

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

7 August - 7 November 1989

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:

Study conducted according to the NTP test protocol, and to GLP. Certain parameters (e.g. haematology, clinical chemistry, urinalysis, immunology and neuropathology) were not assessed.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: NTP 14-week study test method

Deviations:

no

Principles of method if other than guideline:

As part of the NTP investigation into the toxicology of sodium nitrite, 14-week and 2-year studies were conducted in both rats and mice, each differing in the extent of examination. In the 14-week mouse study, a number of routine examinations (e.g. haematology, clinical chemistry, urinalysis) were omitted from the assessment. Certain of these parameters were assessed elsewhere in the overall investigation (14-wk study in rats); an extensive histopathological assessment was conducted.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data, though B6C3F1 mice are often used by the NTP in repeated dose studies

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:

water

Details on oral exposure:

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

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: not applicable
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies of a 0.075 mg/mL dose formulation were performed by the analytical chemistry laboratory using ultraviolet/visible spectrophotometry by measuring absorbance at 347 nm of an aliquot of the sample treated with a salt solution (sodium sulfate and sodium acetate) and a colour reagent (hydrochloric acid, resorcinol and zinconyl chloride). Stability was confirmed for at least 35 days for dose formulations stored at 5oC or at room temperature in the dark.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

Continuously

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 650 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

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): not applicable
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (not further specified)
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: drinking water consumption was measured daily

OPHTHALMOSCOPIC EXAMINATION: No t specified

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (not further specified)

HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 5000 ppm animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, muscle, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, skin, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. The forestomach, testis and spleen of all remaining mice were also examined

OTHER: Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.

Other examinations:

At the end of the studies, samples were collected for sperm motility or vaginal cytology evaluations from mice in the 0, 375, 1500 and 5000 ppm groups. The left cauda, epididymis, and testis were weighed. The following parameters were evaluated: spermatid heads per gram testis, spermatid heads per testis, spermatid count, motility, and concentration. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess (non)neoplastic lesion prevalence. Unless otherwise specified, a value of k=3 was used in the analysis of site-specific lesions. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected Poly-3 tests were used, and reported P values are one sided. Values of P greater than 0.5 are presented as 1 - P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).

Organ and body weight data (continuous variables) were analysed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Spermatid and epididymal spermatozoal data were analysed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no chemical-related clinical findings; no cyanosis or brownish discoloration was observed.

Mortality:

no mortality observed

Description (incidence):

All mice survived until the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights and growth of 990 mg/kg bw/day males and growth of 650 mg/kg bw/day males were significantly less than those of the controls.

A not statistically-significant reduction in final mean body weights and growth was seen in high-dose females.

It was considered that reduced water consumption may have been responsible for the observed growth effects through decreased feed consumption.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption in males (at and above 345 mg/kg bw/day) was less than that of controls at week 13.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative and absolute spleen weights of males were significantly (P ≤ 0.01) greater than those of the controls at the highest dose level (990 mg/kg bw/day) [relative spleen weight was statistically significantly (P ≤ 0.01) increased in males at 650 mg/kg bw/day]. Males in the highest dose group also displayed statistically significant increases in relative heart, kidney and testes weights.

Relative (and absolute) heart, kidney, liver and spleen weights were also significantly increased in females at the highest tested dose level (1230 mg/kg bw/day). Similarly, females in the 840 mg/kg bw/day group showed increased relative and absolute liver and spleen weights, as well as absolute heart and kidney weights.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous cell hyperplasia of the forestomach was apparent in animals at the highest tested dose (990 and 1230 for males and females, respectively). The incidences of extramedullary hematopoiesis in the spleen of males (650 and 990 mg/kg bw/day) and females (at and above 445 mg/kg bw/day) were significantly greater than those in the control groups. Males exposed at the two highest dose levels also showed significantly increased incidences of degeneration of the testis.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm motility in 990 mg/kg bw/day males was decreased relative to the controls and the estrous cycles of 445 and 1230 mg/kg bw/day females were significantly longer than those of the controls

Details on results:

The brownish discoloration and cyanosis seen in rats were not observed in mice. The authors noted that mice may have a higher erythrocyte methaemoglobin reductase activity than do rats, and this was considered a possible explanation for the observed inter-species differences. Increased relative spleen weights occurred in both sexes at the two highest dose levels, and increased splenic extramedullary hematopoiesis was observed. Since the spleen is an erythropoietically active tissue in adult mice, the observed splenic extramedullary hematopoiesis was considered to be consistent with methaemoglobin formation and tissue hypoxia.

The EFSA Panel considered that "tissue hypoxia due to methaemoglobin formation is a strong signal to elicit extramedullary haematopoiesis". Based on extramedullary haematopoiesis in the spleen consistent with methaemoglobin formation, the Panel identified respective NOAELs of 345 and 240 mg/kg bw/day for males and females.

Dose descriptor:

NOAEL

Effect level:

345 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

This value was established by EFSA following a recent review of the data.

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

This value was established by EFSA following a recent review of the data.

Critical effects observed:

not specified

Conclusions:

In a NTP subchronic oral toxicity study, sodium nitrite was provided to mice (10/sex/group) in the drinking water at 375, 750, 1500, 3000 or 5000 ppm (equivalent to approximate dose levels of 90, 190, 345, 650 or 990 mg/kg bw/day for males and 120, 240, 445, 840 or 1230 mg/kg bw/day for females) for 14 weeks. Based on an increased incidence of extramedullary haematopoiesis in the spleen of both sexes, EFSA established NOAELs of 345 and 240 mg/kg bw/day for males and females respectively.

Executive summary:

In a NTP subchronic oral toxicity study, conducted according to the NTP test protocol and to GLP, sodium nitrite was provided to B6C3F1 mice (10/sex/group) in the drinking water at 375, 750, 1500, 3000 or 5000 ppm (equivalent to approximate dose levels of 90, 190, 345, 650 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. Parameters evaluated included mortality, clinical observations, body weight, water consumption, sperm motility, vaginal cytology, selected organ weights, gross and histopathologic examination.

 

No clinical signs of toxicity, mortality or gross lesions were apparent following treatment with sodium nitrite. At the highest two dose levels, males displayed reduced growth while organ weights (heart, kidney, liver, spleen [and testis]) tended to show statistically significant increases in both sexes; generally, there were no concurrent adverse histopathological findings. Upon microscopic assessment, squamous cell hyperplasia of the forestomach was observed in animals at the highest tested dose along with increased incidence of testis degeneration in males at and above 650 mg/kg bw/day. Increased incidence of extramedullary haematopoiesis in the spleen was also observed in males at these dose levels and in females from 445 mg/kg bw/day. Reduced sperm motility was evident in 990 mg/kg bw/day males and the estrous cycles of 445 and 1230 mg/kg bw/day females were significantly longer than those of the controls.

 

The EFSA Panel considered the increased incidence of extramedullary haematopoiesis in the spleen, consistent with methaemoglobin formation and tissue hypoxia, to be the critical effect, establishing NOAELs of 345 and 240 mg/kg bw/day for males and females respectively.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium nitrite

EC Number:

231-555-9

EC Name:

Sodium nitrite

Cas Number:

7632-00-0

Molecular formula:

HNO2.Na

IUPAC Name:

sodium nitrite

Test material form:

solid: crystalline

Test animals

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)

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 ppm

Remarks:

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

Doses / concentrations 2

Dose / conc.:

750 ppm

Remarks:

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

Doses / concentrations 3

Dose / conc.:

1 500 ppm

Remarks:

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

Doses / concentrations 4

Dose / conc.:

3 000 ppm

Remarks:

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

Doses / concentrations 5

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

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.