Calcium nitrite

Endpoint summary

Description of key information

No repeated dose toxicity data with calcium nitrite were identified.

 

In NTP subchronic oral toxicity studies, sodium nitrite was provided to rats and mice (10/species/sex/group) in the drinking water at 375-5000 ppm (equivalent to approximate dose levels of 30-310 mg/kg bw/day for male rats, 40-345 mg/kg bw/day for female rats, 90-990 mg/kg bw/day for male mice and 120-1230 mg/kg bw/day for female mice) for 14 weeks (NTP, 2001). In reviewing these studies, EFSA considered the critical effect in rats 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 male and female rats. Based on an increased incidence of extramedullary haematopoiesis in the spleen of mice, EFSA established NOAELs of 345 and 240 mg/kg bw/day for males and females, respectively (EFSA, 2017).

 

In the analogous chronic oral NTP toxicity studies, sodium nitrite was administered daily via drinking water to rats and mice (50/species/sex/group) at 750, 1500 or 3000 ppm (equivalent to approximate dose levels of 35, 70 or 130 mg/kg bw/day for male rats, 40, 80 or 150 mg/kg bw/day for female rats, 60, 120 or 220 mg/kg bw/day for male mice and 45, 90 or 165 mg/kg bw/day for female mice) for 2 years (NTP, 2001). The critical effect in rats 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 male and female rats. No significant treatment-related adverse effects were observed in mice. As such, the EFSA Panel have identified NOAELs of 220 and 165 mg/kg bw/day for male and female mice, respectively (EFSA, 2017).

 

No repeated dose toxicity studies by the inhalation or dermal route were identified, or are required.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

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

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 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)

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

Dose / conc.:

750 ppm

Remarks:

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

Dose / conc.:

1 500 ppm

Remarks:

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

Dose / conc.:

3 000 ppm

Remarks:

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

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

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).

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.

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.

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.

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

Overall, good-quality database which meets REACH Standard Information Requirements.

System:

haematopoietic

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The key events in the mode of action for the induction of methaemoglobinaemia in humans and experimental animals are as follows: oxidation of the ferrous ion (Fe(II)) in haemoglobin to the ferric ion (Fe(III)), resulting in an increased proportion of methaemoglobin in the blood and leading to a concomitant reduction in oxygen transport capacity (EFSA, 2017; Health Canada, 2013; IARC, 2010; OECD, 2005; WHO, 2011).

 

The reduced oxygen transport becomes clinically manifest when methaemoglobin concentrations exceed 10% of normal haemoglobin concentrations; methaemoglobinaemia causes cyanosis and, at higher concentrations, asphyxia. The normal methaemoglobin level is <2% in humans, and <3% in infants under 3 months of age (WHO, 2011). The OECD considered humans to be more sensitive than rats with respect to the reduced oxygen-carrying capacity of methaemoglobin relative to that of haemoglobin (OECD, 2005). Normally, methaemoglobin can be enzymatically converted back to haemoglobin via the action of NADPH-methaemoglobin reductase; lower quantities and activity of this enzyme in human neonates may explain their susceptibility to methaemoglobinaemia (Health Canada, 2013).

 

More recently, an EFSA Panel evaluated the plausibility and human relevance of the proposed mode of action. Increased methaemoglobin levels can be considered a sensitive endpoint (but potentially oversensitive) and a marker for other effects. The Panel used data from animals to derive the reference point to establish the acceptable daily intake (ADI). In doing so, the Panel took into account that changes in methaemoglobin levels occur in animals and humans following nitrite exposure and that methaemoglobinaemia occurs in humans and animals following increases in nitrite plasma levels. The Panel thus considered methaemoglobin formation in rats of human relevance (EFSA, 2017).

Additional information

No relevant human or laboratory animal data were identified for calcium nitrite.

 

The critical adverse effect of exposure to nitrite is methaemoglobinaemia, a condition whereby methaemoglobin is formed in the blood due to the interaction of haemoglobin with nitrite (EFSA, 2017; Health Canada, 2013; IARC, 2010; OECD, 2005; WHO, 2011). Higher concentrations of methaemoglobin result in cyanosis, hypoxia and eventually death (EFSA, 2017).

 

Several oral laboratory animal repeated dose toxicity studies are available for sodium (and potassium) nitrite, and a recent EFSA (2017) report has reviewed these. The key studies are the NTP subchronic and chronic drinking water studies on sodium nitrite in rodents (NTP, 2001). Sodium nitrite is closely related to calcium nitrite, and is considered a suitable surrogate for read-across for this endpoint. [See read-across justification report in IUCLID section 13 for details.]

 

In the subchronic oral toxicity studies, conducted according to the NTP test protocol and to GLP, sodium nitrite was provided to rats (core study) and mice (10/species/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 male rats, 40, 80, 130, 225 or 345 mg/kg bw/day for female rats, 90, 190, 345, 650 or 990 mg/kg bw/day for male mice and 120, 240, 445, 840 or 1230 mg/kg bw/day for female mice) for 14 weeks. Additional clinical pathology study rats (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 mice and core study rats. Clinical pathology groups were assessed for a variety of haematology and clinical chemistry parameters, notably haemoglobin, methaemoglobin and nitrosamine concentrations. In rats, 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. No clinical signs of toxicity, mortality or gross lesions were apparent in mice 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 (NTP, 2001).In reviewing these studies in detail, the EFSA Panel considered the methaemoglobin increases (with associated effects on reticulocyte count and mean cell volume/haemoglobin concentration) to be the critical effect in rats, establishing respective NOAELs of 115 and 130 mg/kg bw/day for males and females. In mice, the 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 (EFSA, 2017).

 

In the analogous chronic oral toxicity studies, conducted according to the NTP test protocol and to GLP, sodium nitrite was provided in the drinking water to core study F344/N rats and B6C3F1 mice (50/species/sex/group) at 750, 1500 or 3000 ppm (equivalent to approximate dose levels of 35, 70 or 130 mg/kg bw/day for male rats, 40, 80 or 150 mg/kg bw/day for female rats, 60, 120 or 220 mg/kg bw/day for male mice and 45, 90 or 165 mg/kg bw/day for female mice) for 2 years. Additional special study animals (10/species/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 of rats. 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. Sodium nitrite treatment of mice 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 (NTP, 2001). The EFSA Panel considered the methaemoglobin increases (statistically significant at the highest tested dose level) to be the critical effect in rats, establishing NOAELs of 70 and 80 mg/kg bw/day for males and females, respectively. In mice, the 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 (EFSA, 2017).

The observed effects in the various repeated dose NTP investigations were generally more severe in rats than in mice. The NTP investigators 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 (NTP, 2001).

 

The EFSA Panel considered that the short-term, subchronic and chronic toxicity studies in rats and mice using sodium and potassium nitrite [including the NTP investigations on sodium nitrite] primarily confirmed that the main observed effect is the formation of methaemoglobin. Additional effects reported during exposure to sodium nitrite (including increased erythropoietic activity and changes in haematological parameters) were considered by the Panel as secondary to the formation of methaemoglobin and to a reduced capacity to transport oxygen to tissues. Other reported effects included reduced blood pressure in rats and humans, as well as vasodilation effects in humans. Overall, none of those additional effects could be considered as a basis on which to establish an

ADI due to the lack of precise details on the exact doses tested, the lack of precise classification of

lesions reported, of a plausible mechanism of action, too large dose-spacing and the lack of full dose relationships. The Panel selected the subchronic NTP study (2001) in rats as a key study because five doses had methaemoglobin levels higher than the control (cf. one dose in the other studies, rendering them unsuitable for the derivation of a health-based guidance value) (EFSA, 2017).

 

In addition, according to a NTP (1990) report citing the National Research Council (NRC, 1977), an early “3-generation lifetime study found no evidence of “chronic toxicity, carcinogenicity or teratogenicity” in rats receiving about 100 mg/kg bw/day sodium nitrite from the drinking water (Druckery et al., 1963). [No further details of this early study provided in the citing NTP report.]

 

 

References (not included in IUCLID ESRs)

 

Druckery H, Steinhoff D, Beuthner H, Schneider H and Klarner P (1963). Prufung von nitrit auf chronisch toxische wirkung und ratten. Arzneimihelforschung 13, 320-323 [cited in NRC, 1977.]

 

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

 

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

 

NRC (1977). National Research Council. [No title given.] Drinking Water and Health Volume 1. p 420 [cited in NTP, 1990.]

 

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

Justification for classification or non-classification

In reviewing the repeated dose toxicity (oral) NTP studies, the EFSA Panel considered the NOAELs of 115 and 130 mg/kg bw/day for male and female rats in the 90-day oral studies as critical. As such, classification of this substance as STOT-RE is not required, according to EU CLP criteria (EC 1272/2008).