Calcium nitrite

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 August - 10 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. urinalysis, immunology and neuropathology) were not assessed.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other 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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 August 1995 - 15 August 1997

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. 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 2-year 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 2-year rat study, a number of routine examinations (e.g. haematology, clinical chemistry, urinalysis, organ weights) were omitted from the assessment. Certain of these parameters were assessed elsewhere in the overall investigation (14-wk studies in rats and/or mice).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: F344/N

Details on species / strain selection:

No data, though F344/N rats 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 Laboratory Animals and Services (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 (animals/cage: core study, 2 or 3 (males) or 5 (females); special study, 2 or 3 (males) or 5 (females); aged sentinel animal study, 3), changed twice weekly; rotated every 2 weeks
- Diet (e.g. ad libitum): NTP-2000 pelleted diet, irradiated beginning 22 July 1996, available ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water, available ad libitum and changed twice weekly
- Acclimation period: 11 days (males) or 12 days (females)

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: 14 August 1995 (males) and 15 August 1995 (females) To: 11-13 August 1997 (males) and 13-15 August 1997 (females)

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared approximately every 4 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 color 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:

105 weeks

Frequency of treatment:

Continuously

Dose / conc.:

750 ppm

Remarks:

Equivalent to approximately 35 mg/kg bw/day (males) and 40 mg/kg bw/day (females).

Dose / conc.:

1 500 ppm

Remarks:

Equivalent to approximately 70 mg/kg bw/day (males) and 80 mg/kg bw/day (females).

Dose / conc.:

3 000 ppm

Remarks:

Equivalent to approximately 130 mg/kg bw/day (males) and 150 mg/kg bw/day (females).

No. of animals per sex per dose:

50/sex/dose in core study [10/sex/dose for special study and 15/sex/dose for aged sentinel study]

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of the 14-week study
- Rationale for animal assignment (if not random): not applicable
- Rationale for selecting satellite groups: toxicokinetic assessment of plasma nitrite and blood methemoglobin (special study; groups of 10 rats/sex/dose were exposed to the same concentrations as in the core study for 12 months), (aged sentinel study; groups of 15 rats/sex received a single gavage dose of 40 mg/kg bw after 18 months).
- Post-exposure recovery period in satellite groups: none
- 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: days 8 and 36, and then at 4-week intervals thereafter until necropsy (core study animals)

BODY WEIGHT: Yes
- Time schedule for examinations: days 8 and 36, and then at 4-week intervals thereafter until necropsy (core study animals); 2 weeks and 3 months (special study animals); 18 months (aged sentinel study animals)

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: over a 1-week period at 4-week intervals (core study animals)

OPHTHALMOSCOPIC EXAMINATION: Not examined

HAEMATOLOGY: No [conducted during 14-week study]

CLINICAL CHEMISTRY: No [conducted during 14-week study]

URINALYSIS: Not examined

NEUROBEHAVIOURAL EXAMINATION: Not examined

IMMUNOLOGY: Not examined

OTHER: Blood samples were collected from the retroorbital sinus of special study animals (10/sex/group) at 2 weeks and 3 months and from aged sentinel animals (15/sex/group) at 18 months for determination of plasma nitrite and blood methaemoglobin concentrations. [For special study animals, blood was collected from two animals per group per time point (06:00, 12:00, 21:00, 24:00, and 03:00 hours). Two or three aged sentinel animals were sampled at each time point (2, 5, 10, 30, or 60 minutes after gavage dosing).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Necropsies were performed on all core study animals and certain aged sentinel rats (5/sex). At necropsy, all organs and tissues were examined for grossly visible lesions. For all paired organs (i.e., adrenal gland, kidney, ovary), samples from each organ were examined.

HISTOPATHOLOGY: Yes. Complete histopathology was performed on all core study 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, 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.

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).

Body weight data (continuous variable) was analysed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Blood/plasma concentration and toxicokinetic 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 clinical findings related to exposure to sodium nitrite; the brown discoloration and cyanosis seen in the 14-week study were not observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival of exposed groups was similar to that of the controls (29/50, 38/50, 36/50 and 36/50 for males at doses of 0, 35, 70 and 130 mg/kg bw/day respectively, and 33/50, 31/50, 36/50 and 33/50 for females at 0, 40, 80 or 150 mg/kg bw/day respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of high-dose animals (130 and 150 mg/kg bw/day for males and females, respectively) were less than those of the controls (6-10%) throughout the study; no statistical significance was reported.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption by high dose males (130 mg/kg bw/day) and females (150 mg/kg bw/day) was less than that of the controls throughout the study. Other exposed groups generally had lower water consumption from week 14..

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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidence of mammary gland fibroadenoma was increased in all treated females (significant only at 80 mg/kg bw/day); the incidences in controls were equal to the highest incidence in the historical control database. When combined with adenomas, no statistically significant increases were apparent and the incidences of carcinoma were not increased in the exposed groups. The incidences of multiple fibroadenoma were increased in females exposed at 40 and 80 mg/kg bw/day. However, fibroadenomas are the most common benign neoplasms that occur in the mammary gland of this species of rat and the NTP investigators noted that, unlike benign neoplasms in other tissues that usually progress to malignancy, fibroadenomas are generally considered to represent an end-stage lesion, and progression to carcinoma is rare. (The EFSA Panel agreed.)

Although the incidence of skin fibroma was significantly increased in males at 70 mg/kg bw/day, this was considered unrelated to treatment on the basis of a lack of a dose response, the lack of a significant increase in the incidences of fibrosarcomas of the skin, the fact that their combined incidences were within the historical range for NTP controls, and that they are the most common neoplams that occur in the skin of F344/N rats.

The incidences of mononuclear cell leukemia (MCL) were significantly decreased in males and females at the highest two dose levels and were less than the historical ranges for NTP controls. These findings indicate that sodium nitrite reduced MCL incidence in rats, thereby resulting in increased survival.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the forestomach, the incidence of hyperplasia of the squamous epithelium was statistically significantly increased at the highest tested dose (130 and 150 for males and females, respectively).

There was a significant increase in the incidence of chronic active inflammation of the liver in males at 70 and 130 mg/kg bw/day though this, being a common spontaneous lesion in F344/N rats, was not considered to be related to sodium nitrite exposure. Similarly, nephropathy was marginally increased in high-dose females; the NTP investigators were unclear whether this was treatment related since this effect is also a common spontaneous lesion in this species.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically, the mammary gland fibroadenomas observed in exposed females were similar to those in the controls. No forestomach neoplasms were observed following exposure to sodium nitrite.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At both 2 weeks and three months, methaemoglobin levels were high at night when the rats were actively feeding and drinking and low during the day when the rats were less active. Methaemoglobin levels tended to increase with increasing dosage. The EFSA Panel noted that "blood methaemoglobin
concentrations only differ statistically between controls and the 3,000 mg/L treated animals", though statistically significant differences were also observed at select blood sampling time points for mid-dose animals.

Details on results:

The reduced body weights were probably related to lower water consumption as in the 14-week study.

The increased methaemoglobin concentrations and lower body weights of the exposed rats may be factors in the reduced MCL incidences.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

Remarks on result:

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Remarks on result:

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

Critical effects observed:

not specified

Conclusions:

In an oral carcinogenicity study, sodium nitrite was administered daily via drinking water to rats (50/sex/group) at 750, 1500 or 3000 ppm (equivalent to approximate dose levels of 35, 70 or 130 mg/kg bw/day for males and 40, 80 or 150 mg/kg bw/day for females) for 105 weeks. The critical effect was a dose-dependent increase in methaemoglobin concentration (considered by EFSA to be statistically significant at the highest tested dose level). On this basis, EFSA established respective NOAELs of 70 and 80 mg/kg bw/day for males and females.

Executive summary:

In an oral carcinogenicity study, conducted according to the NTP test protocol and to GLP, sodium nitrite was provided in the drinking water to core study F344/N rats (50/sex/group) at 750, 1500 or 3000 ppm (equivalent to approximate dose levels of 35, 70 or 130 mg/kg bw/day for males and 40, 80 or 150 mg/kg bw/day for females) for 105 weeks. Additional special study animals (10/sex/group) were similarly treated with sodium nitrite for 12 months for toxicokinetic studies. Control animals received vehicle only. Parameters evaluated included mortality, clinical observations, body weight, water consumption, gross and histopathologic examination of a wide-range of organs and tissues for all core study animals. Blood was collected from special study groups at 2 weeks and 3 months for the determination of plasma nitrite and methaemoglobin concentrations.

A dose-dependent increase in methaemoglobin levels (compared to the controls) was observed in both sexes. Growth was reduced (although not statistically significantly) at the highest tested dose level, possibly due to lower water consumption. Sodium nitrite reduced the incidence of mononuclear cell leukaemia, resulting in increased survival of treated rats. The incidence of forestomach hyperplasia was statistically significantly increased at the highest tested dose (but no forestomach neoplasms seen). Increased incidences of mammary gland fibroadenoma (all treated females) and skin fibroma (mid-dose males) were considered unrelated to treatment. Similarly, the increase in chronic active inflammation of the liver in males at and above 70 mg/kg bw/day was also discounted. Although nephropathy was marginally increased in high-dose females, as a common spontaneous lesion in this species, the relevance of this effect was unclear. 

The EFSA Panel considered the methaemoglobin increases (statistically significant at the highest tested dose level) to be the critical effect, establishing NOAELs of 70 and 80 mg/kg bw/day for males and females, respectively.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2 August 1995 - 1 August 1997

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 2-year 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 2-year mouse study, a number of routine examinations (e.g. haematology, clinical chemistry, urinalysis, organ weights) were omitted from the assessment. Certain of these parameters were assessed elsewhere in the overall investigation (14-wk studies in rats and/or mice).

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 Laboratory Animals and Services (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 (animals/cage: core and special studies, 1 (males) or 5 (females); aged sentinel animal study, 1 (male) or 3 (females)), changed weekly (males) or twice weekly (females); rotated every 2 weeks
- Diet (e.g. ad libitum): NTP-2000 pelleted diet, irradiated beginning 16 July 1996, available ad libitum, changed weekly (males) or twice weekly (females)
- Water (e.g. ad libitum): Tap water, available ad libitum and changed weekly (males) or twice weekly (females)
- Acclimation period: 13 days (males) or 14 days (females)

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: 2 August 1995 (males) and 3 August 1995 (females) To: 28-30 July 1997 (males) and 30 July - 1 August 1997 (females)

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared approximately every 4 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 color 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:

104-105 weeks

Frequency of treatment:

Continuously

Dose / conc.:

750 ppm

Remarks:

Equivalent to approximately 60 mg/kg bw/day (males) and 45 mg/kg bw/day (females).

Dose / conc.:

1 500 ppm

Remarks:

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

Dose / conc.:

3 000 ppm

Remarks:

Equivalent to approximately 220 mg/kg bw/day (males) and 165 mg/kg bw/day (females).

No. of animals per sex per dose:

50/sex/dose in core study [10/sex/dose for special study and 15/sex/dose for aged sentinel study]

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of the 14-week study
- Rationale for animal assignment (if not random): not applicable
- Rationale for selecting satellite groups: toxicokinetic assessment of plasma nitrite and blood methaemoglobin (special study; groups of 10 mice/sex/dose were exposed to the same concentrations as in the core study for 12 months), (aged sentinel study; groups of 15 mice/sex received a single gavage dose of 40 mg/kg bw after 18 months).
- Post-exposure recovery period in satellite groups: none
- 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: days 8 and 36, and then at 4-week intervals thereafter until necropsy (core study animals)

BODY WEIGHT: Yes
- Time schedule for examinations: days 8 and 36, and then at 4-week intervals thereafter until necropsy (core study animals); 12 months (special study animals); 18 months (aged sentinel study animals)

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: over a 1-week period at 4-week intervals (core study animals)

OPHTHALMOSCOPIC EXAMINATION: Not examined

HAEMATOLOGY: Not examined

CLINICAL CHEMISTRY: Not examined

URINALYSIS: Not examined

NEUROBEHAVIOURAL EXAMINATION: Not examined

IMMUNOLOGY: Not examined

OTHER: Blood samples were collected from the retroorbital sinus of special study animals (10/sex/group) at 12 months and from aged sentinel animals (15/sex/group) at 18 months for determination of plasma nitrite and blood methaemoglobin concentrations. [For special study animals, blood was collected from two animals per group per time point (06:00, 12:00, 21:00, 24:00, and 03:00 hours). Two or three aged sentinel animals were sampled at each time point (2, 5, 10, 30, or 60 minutes after gavage dosing)].

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Necropsies were performed on all core study animals and certain aged sentinel mice (5/sex). At necropsy, all organs and tissues were examined for grossly visible lesions. For all paired organs (i.e., adrenal gland, kidney, ovary), samples from each organ were examined.

HISTOPATHOLOGY: Yes. Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (females only), 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.

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).

Body weight data (continuous variable) was analysed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Blood/plasma concentration and toxicokinetic 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 clinical findings related to exposure to sodium nitrite.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival of exposed groups was similar to that of the controls (39/50, 45/50, 42/50 and 39/50 for males at doses of 0, 60, 120 or 220 mg/kg bw/day respectively, and 40/50, 34/50, 37/50 and 41/50 for females at 0, 45, 90 or 165 mg/kg bw/day respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of exposed groups were generally similar to those of the controls throughout the study, except for those of high-dose females which were consistently less than those of the controls (though not at the end of the study); no statistical significance was reported.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption by the exposed groups was generally less than that of the controls throughout the study.

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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was "slightly" increased in all treated females but was within the historical control range; these increased incidences were not statitically significant or exposure concentration related and were not accompanied by any associated preneoplastic lesions and were not considered to be related to sodium nitrite exposure.

Although the incidence of skin fibrosarcoma was significantly increased in females at 45 mg/kg bw/day (also exceeding the historical control range), this was considered unrelated to treatment on the basis of a lack of a dose response and the fact that the combined incidence of fibroma or fibrosarcoma was within the historical range for NTP controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in the incidence of epithelial hyperplasia of the glandular stomach was observed in males at the highest tested dose (220 mg/kg bw/day).

In the forestomach, the incidence of hyperplasia of the squamous epithelium was increased in females at the highest tested dose (165 mg/kg bw/day).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In females, the incidences of squamous cell papilloma or carcinoma (combined) of the forestomach showed a positive trend, and the incidence in high-dose animals (165 mg/kg bw/day) exceeded the default historical control range for the study; incidence was also increased when compared to a more extensive control database (including additional exposure routes), with the exception of corn oil gavage studies. These findings were not statistically significant.

No forestomach neoplasms were observed in males and there were no glandular stomach neoplasms in either sex following exposure to sodium nitrite.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At 12 months, no nitrite was detected in the plasma of control or low-dose males or in any group of females. In general, there was an exposure concentration-related increase in plasma nitrite in males at 120 and 220 mg/kg bw/day; peak plasma nitrite concentrations occurred around midnight. Blood methaemoglobin concentrations were similar among exposed groups of males and females. (The EFSA Panel agreed.)

Details on results:

Females displayed a positive trend in the incidences of squamous cell papilloma or carcinoma (combined) in the forestomach which may have been related to exposure to sodium nitrite. Although the incidence at the highest tested dose (165 mg/kg bw/day) exceeded the historical control range for forestomach neoplasms no statistical significance was achieved, though the finding of two squamous cell carcinomas was supportive of a chemical-related effect because forestomach carcinomas are rarely observed in controls in drinking water studies. Increased incidences of forestomach neoplasms were not observed in exposed males (or in the analogous study in rats), though the forestomach was clearly a target organ because hyperplasia was observed in both species in the 14-week studies and in rats in the 2-year study. Interestingly, although forestomach hyperplasia was not observed in male mice in the 2-year study, hyperplasia of the glandular epithelium of the stomach was seen at the highest tested dose.

Proliferative lesions involving the squamous epithelium represent a continuum, progressing from focal hyperplasia to papilloma to squamous cell carcinoma. Hyperplasia was generally mild. In carcinomas, there was a focal invasion of the squamous epithelium into the lamina propria, though there was no infiltration of neoplastic cells through the serosa of the forestomach, and there was no metastasis.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no statistically significant treatment-related effects

Remarks on result:

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no statistically significant treatment-related effects

Remarks on result:

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

Critical effects observed:

not specified

Conclusions:

In an oral carcinogenicity study, mice (50/sex/group) were provided with sodium nitrite in the drinking water at 750, 1500 or 3000 ppm (equivalent to approximate dose levels of 60, 120 or 220 mg/kg bw/day for males and 45, 90 or 165 mg/kg bw/day for females) for 104-105 weeks. No significant treatment-related adverse effects were observed. As such, the EFSA Panel have identified NOAELs of 220 and 165 mg/kg bw/day for males and females, respectively.

Executive summary:

In an oral carcinogenicity study, conducted according to the NTP test protocol and to GLP, sodium nitrite was provided in the drinking water to core study B6C3F1 mice (50/sex/group) at 750, 1500 or 3000 ppm (equivalent to approximate dose levels of 60, 120 or 220 mg/kg bw/day for males and 45, 90 or 165 mg/kg bw/day for females) for 104-105 weeks. Additional special study animals (10/sex/group) were similarly treated with sodium nitrite for 12 months for toxicokinetic studies. Control animals received vehicle only. Parameters evaluated included mortality, clinical observations, body weight, water consumption, gross and histopathologic examination of a wide-range of organs and tissues for all core study animals. Blood was collected from special study groups at 12 months for the determination of plasma nitrite and methaemoglobin concentrations.

Sodium nitrite treatment did not influence survival rates or clinical findings, while blood methaemoglobin concentrations did not differ statistically among treated and control groups. Mean body weights of high-dose females were generally lower than those of controls (although not statistically significantly) at the highest tested dose level, possibly due to lower water consumption. In females, there was a positive trend in the incidences of squamous cell papilloma or carcinoma (combined) in the forestomach, exceeding the historical control range at the highest tested dose (165 mg/kg bw/day); no statistical significance was achieved. Incidence of hyperplasia, a pre-neoplastic lesion, in this tissue was also elevated in high-dose females. While there was no such effect in males, a statistically significant increase in the incidence of epithelial hyperplasia of the glandular stomach was observed at the highest tested dose (220 mg/kg bw/day), though no neoplasms were seen. Increased incidences of alveolar/bronchiolar adenoma or carcinoma (combined) (all treated females) and skin fibrosarcoma (low-dose females) were considered unrelated to treatment.

The EFSA Panel evidently did not consider the increased glandular stomach hyperplasia in males (statistically significant at the highest tested dose level) to be a critical effect, establishing NOAELs of 220 and 165 mg/kg bw/day for males and females, respectively.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2001

Report date:

2001

Reference 2

Reference Type:

review article or handbook

Title:

Re-evaluation of potassium nitrite (E 249) and sodium nitrite (E 250) as food additives.

Author:

EFSA

Year:

2017

Bibliographic source:

EFSA Journal 15(6):4786

Report date:

2017

Materials and methods

Test guideline

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 rat study, urinalysis (a routine examination) was omitted from the assessment, though an extensive histopathological assessment was conducted.

GLP compliance:

yes

Limit test:

no

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:

rat

Strain:

other: F344/N

Details on species / strain selection:

No data, though F344/N rats 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: 7 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 (5 animals/cage), changed twice 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: 14 days (males) or 15 days (females)

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: 10 August 1989 (males) and 11 August 1989 (females) To: 9 November 1989 (males) and 10 November 1989 (females)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 ppm

Remarks:

Equivalent to approximately 30 mg/kg bw/day (males) and 40 mg/kg bw/day (females).

Doses / concentrations 2

Dose / conc.:

750 ppm

Remarks:

Equivalent to approximately 55 mg/kg bw/day (males) and 80 mg/kg bw/day (females).

Doses / concentrations 3

Dose / conc.:

1 500 ppm

Remarks:

Equivalent to approximately 115 mg/kg bw/day (males) and 130 mg/kg bw/day (females).

Doses / concentrations 4

Dose / conc.:

3 000 ppm

Remarks:

Equivalent to approximately 200 mg/kg bw/day (males) and 225 mg/kg bw/day (females).

Doses / concentrations 5

Dose / conc.:

5 000 ppm

Remarks:

Equivalent to approximately 310 mg/kg bw/day (males) and 345 mg/kg bw/day (females).

No. of animals per sex per dose:

10 [15 for clinical pathology study]

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: assessment of clinical pathology (study groups of 15 males and 15 females were exposed to the same concentrations as in the core study for 70 or 71 days.
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): no data

Positive control:

none

Examinations

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (core study animals)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (core study animals)

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: Yes
- Time schedule for collection of blood: Days 5 and 19 for clinical pathology study rats and from core study rats at the end of the study.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 10
- Parameters examined.: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials; erythrocyte and platelet morphologic assessments; methemoglobin concentration; reduced glutathione concentration in erythrocytes; and Heinz body count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days 5 and 19 for clinical pathology study rats and from core study rats at the end of the study.
- Animals fasted: No
- How many animals: 10
- Parameters examined.: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

OTHER: Blood samples were collected from the abdominal aorta of clinical pathology study rats (5/sex/group) on day 70 or 71 for analysis of haemoglobin, methaemoglobin and nitrosamine concentrations; stomach contents were also collected for nitrosamine concentrations.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (not further specified)

HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 5000 ppm core study 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 of 750 (males), 1500, and 3000 ppm rats was 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 rats in the 0, 375, 750 (females only) 1500 (males only), 3000 (females only) and 5000 ppm (males only) 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). Clinical pathology, 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).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown discoloration in the eyes and cyanosis of the mouth, tongue, ears, and feet of males exposed at 200 and 310 mg/kg bw/day and of females exposed at and above 130 mg/kg bw/day.

Males in the 310 mg/kg bw/day group were hypoactive, and a few females exposed at and above 40 mg/kg bw/day developed alopecia.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female died during week 4 in the 225 mg/kg bw/day group. No other mortality occurred and no dose-related trend was apparent.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights and growth of 200 and 310 mg/kg bw/day males and 345 mg/kg bw/day females were significantly less than those of the controls [P ≤ 0.05 in 225 mg/kg bw/day 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 (310 mg/kg bw/day) and females (225 and 345 mg/kg bw/day) was (much) less than that of controls at weeks 2 and 14.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Methaemoglobin levels were significantly elevated in all treated groups compared to the controls by the end of the treatment period, and a dose-response relationship was seen. Reticulocyte counts were increased in 200 and 310 mg/kg bw/day males and 345 mg/kg bw/day females; mean cell volumes and mean cell haemoglobin values were increased in males (at 200 mg/kg bw/day) and females (225 and 345 mg/kg bw/day). The erythron was decreased on day 19 but increased by week 14 in males and females at the highest tested dose (310 and 345 mg/kg bw/day, respectively). Transient effects on various other haematological parameters were also observed, though these normalised by the end of the study.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increases in urea nitrogen concentrations occurred on day 19 in high-dose males and females and at week 14 in all exposed groups of males and at or above 130 mg/kg bw/day in females. Tranisent increases in creatinine concentrations, another marker of renal function, were observed in females, though these normalised by the end fo the study; male rats were unaffected. On day 5, there was a decrease in the serum activity of alkaline phosphatase in various exposed groups, though this effect had reversed by the end of the study in most groups.

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 kidney weights of males and females were significantly (P ≤ 0.01) greater than those of the controls at the two highest dose levels (200 and 310 mg/kg bw/day males and 225 and 345 mg/kg bw/day females) [relative kidney weight was statistically significantly (P ≤ 0.05) increased in males at all doses]. Relative (and absolute) spleen weights were also significantly (P ≤ 0.01) increased in both sexes at these dose levels [P ≤ 0.05 in males at 115 mg/kg bw/day]. At the two highest dose levels, relative heart weight was significantly (P ≤ 0.01) increased in both sexes [P ≤ 0.05 in females at 225 mg/kg bw/day].

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased incidence of erythropoietic activity was observed in males (5/10, 5/10, 5/10, 8/10, 8/10, 9/10) and females (1/10, 0/10, 1/10, 3/10, 7/10, 10/10); these were considered to be consistent with the haematologic findings of regenerative anemia. There were no abnormal changes seen in the kidney or spleen. Squamous cell hyperplasia of the forestomach was apparent in all animals at the highest tested dose (310 and 345 for males and females, respectively).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm motility in 115 and 310 mg/kg bw/day males was significantly decreased relative to the controls. There were no significant differences in vaginal cytology parameters between exposed and control females.

N-Nitrosodimethylamine and N-nitrosopyrrolidine were not detected in the blood of treated groups (levels of detection were less than 2.0 ppb for the highest level of sensitivity). Total nitrosamine concentrations in the blood of exposed males and females were not significantly different from control values. N-Nitrosodimethylamine and N-nitrosopyrrolidine were not detected in the stomach contents of exposed males or females.

Details on results:

The increased mean cell volume/haemoglobin values observed in week 14 are consistent with reticulocytosis. However, the authors noted that these values should also have increased in affected animals on days 5 and 19. This did not happen, and the values for the 5000 ppm animals decreased on day 19. This suggests that more than one mechanism affected red blood cell size, that the factor(s) resulting in small cells had more influence at the early time points, and that this effect abated with time.

The magnitude of methemoglobinemia remained fairly constant for the 5000 ppm groups, though the magnitude of the erythropoietic response appeared to diminish with time. The mechanism for this amelioration is unknown but may reflect an acclimation of the animals to lower tissue oxygen concentrations.

The mechanism for the transient increase in platelet counts that occurred in 5000 ppm animals is unknown, but it was possible that nitrite administration altered nitric oxide metabolism which may have contributed to the platelet effects.

The EFSA Panel considered the methaemoglobin increases to be a relevant effect, and used this data to conduct benchmark dose (BMD) modelling. Methaemoglobin levels were measured at day 5, day 19 and week 14 and BMD modelling was performed for every time point. Only the data from week 14 resulted in acceptable modelling. Lower bound BMD (BMDL) values of 9.63 and 14.62 mg/kg bw/day were identified for males and females respectively. The Panel also noted that although high levels of methaemoglobin are directly adverse, lower levels should be regarded as either precursors of such direct adversity or as markers of exposure which increase prior to clinical manifestation of adverse effects. The use of more sensitive markers as a basis for determining a reference point which ensures adversity does not occur is a long established approach (e.g. preneoplastic lesions) which is more protective than using adversity per se.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

115 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

Remarks on result:

other:

Remarks:

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

2

Dose descriptor:

NOAEL

Effect level:

130 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Remarks on result:

other:

Remarks:

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a NTP subchronic oral toxicity study, sodium nitrite was provided to rats (10/sex/group) in the drinking water at 375, 750, 1500, 3000 or 5000 ppm (equivalent to approximate dose levels of 30, 55, 115, 200 or 310 mg/kg bw/day for males and 40, 80, 130, 225 or 345 mg/kg bw/day for females) for 14 weeks. EFSA considered the critical effect to be a dose-dependent increase in methaemoglobin concentration (with concomitant increases in reticulocyte count and mean cell volume/haemoglobin concentration at the two highest dose levels). On this basis, EFSA established respective NOAELs of 115 and 130 mg/kg bw/day for males and females.

Executive summary:

In a subchronic oral toxicity study, conducted according to the NTP test protocol and to GLP, sodium nitrite was provided to core study rats (10/sex/group) in the drinking water at 375, 750, 1500, 3000 or 5000 ppm (equivalent to approximate dose levels of 30, 55, 115, 200 or 310 mg/kg bw/day for males and 40, 80, 130, 225 or 345 mg/kg bw/day for females) for 14 weeks. Additional clinical pathology study animals (15/sex/group) were similarly treated with sodium nitrite for around 10 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 for all core study animals. Clinical pathology groups were assessed for a variety of haematology and clinical chemistry parameters, notably haemoglobin, methaemoglobin and nitrosamine concentrations.

 

Methaemoglobin levels (compared to the controls) were significantly increased in all treated groups at the end of the treatment period, and a dose-response relationship was seen. A concomitant increase in reticulocyte count, mean cell volume and mean haemoglobin concentration was observed at the two highest dose levels. Statistically significant reduced growth and increased relative organ weights (heart, kidney and spleen) were also essentially limited to these levels in both sexes, though there were no concurrent adverse histopathological findings. No dose-related trend in mortality was apparent and there were no gross lesions. Microscopic assessment revealed squamous cell hyperplasia of the forestomach in all animals at the highest tested dose along with increased erythropoietic activity in the bone marrow of both sexes, consistent with the haematologic findings of regenerative anaemia. The effects on various clinical chemistry parameters are of limited relevance given the lack of histopathological findings in the major organs. Treated females displayed no effects on vaginal cytology parameters, though sperm motility was reduced in 115 and 310 mg/kg bw/day males.

 

The EFSA Panel considered the methaemoglobin increases (with associated effects on reticulocyte count and mean cell volume/haemoglobin concentration) to be the critical effect, establishing respective NOAELs of 115 and 130 mg/kg bw/day for males and females.