Calcium nitrite

Carcinogenicity

Description of key information

No carcinogenicity data with calcium nitrite were identified.

 

According to a recent EFSA (2017) review, no evidence of carcinogenic activity was seen in NTP rodent cancer bioassays in which rats and mice were exposed to sodium nitrite in the drinking water at up to 3000 ppm for about 2 years (NTP, 2001). 

 

No carcinogenicity studies by the inhalation or dermal route were identified, or are required.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: 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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: NTP 2-year study test method

Deviations:

no

GLP compliance:

yes

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) carcinogenicity studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: no data
- 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)

DETAILS OF FOOD AND WATER QUALITY: no data

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 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

Post exposure period:

none

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:

- Toxicokinetic data : yes (plasma nitrite and blood methaemoglobin)
- 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 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.

Dermal irritation (if dermal study):

not examined

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 middose 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.

Relevance of carcinogenic effects / potential:

The NTP investigators concluded that there was "no evidence of carcinogenic activity" in this study. (The EFSA Panel agreed with this conclusion.)

Dose descriptor:

NOAEL

Effect level:

130 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no evidence of carcinogenicity at any dose level

Remarks on result:

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

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no evidence of carcinogenicity at any dose level

Remarks on result:

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

Critical effects observed:

not specified

Conclusions:

No evidence of carcinogenicity was observed in a 2-year NTP study involving daily exposure of rats to sodium nitrite via drinking water at up to 3000 ppm. On this basis, EFSA established carcinogenicity NOAELs of 130 and 150 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. Gross and histopathologic examination of a wide-range of organs and tissues was conducted for all core study animals.

 

No evidence of carcinogenic activity was observed in this study. 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 were seen). Increased incidences of mammary gland fibroadenoma (all treated females) and skin fibroma (mid-dose males) were considered unrelated to treatment. There were no additional neoplastic findings.

 

The EFSA Panel concluded that there was no evidence of carcinogenic activity in this study, establishing carcinogenicity NOAELs of 130 and 150 mg/kg bw/day for males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

130 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

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

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data identified.

Justification for classification or non-classification

No carcinogenicity studies with calcium nitrite were identified. According to a recent EFSA (2017) review, no evidence of carcinogenic activity was seen in reliable NTP rodent cancer bioassays with sodium nitrite. As such, classification of calcium nitrite for carcinogenicity is not required, according to EU CLP criteria (EC 1272/2008).

Additional information

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

 

Several laboratory animal carcinogenicity studies are available for sodium nitrite, and a recent EFSA (2017) report has reviewed these. The key studies are the rodent 2-year NTP drinking water studies (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 first, 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. Gross and histopathologic examination of a wide-range of organs and tissues was conducted for all core study animals. No evidence of carcinogenic activity was observed in this study. 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 were seen). Increased incidences of mammary gland fibroadenoma (all treated females) and skin fibroma (mid-dose males) were considered unrelated to treatment. There were no additional neoplastic findings (NTP, 2001). The EFSA Panel concluded that there was no evidence of carcinogenic activity in this study, establishing carcinogenicity NOAELs of 130 and 150 mg/kg bw/day (the highest tested doses) for male and female rats respectively (EFSA, 2017).

 

In the second, 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. Control animals received vehicle only. Gross and histopathologic examination of a wide-range of organs and tissues was conducted for all core study animals. 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 did not consider the positive trend in forestomach tumour incidence in females (not statistically significant at any dose level) to be a critical effect, establishing carcinogenicity NOAELs of 220 and 165 mg/kg bw/day (the highest tested doses) for male and female mice respectively (EFSA, 2017).

 

In a 2-year water drinking study in rats, no treatment-related increase in tumours of any tissue examined up to a dose of 125 mg/kg bw/day was reported (Maekawa et al., 1982).

Overall, the EFSA Panel considered that the available carcinogenicity studies in mice and rats, generally meeting present requirements for toxicity testing, did not show evidence of carcinogenic potential for sodium nitrite (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.]

 

The EFSA Panel noted that the potential carcinogenicity of nitrite in humans has been extensively reviewed by IARC (2010). The overall conclusions of the Panel are based on the IARC evaluation and on the evaluation of epidemiological studies published subsequently. The Panel concluded that there was evidence that the intake of preformed N-nitrosodimethylamine (NDMA) was associated with increased risk of colorectal cancer (CRC) or its subtypes and, there was some evidence to link the combination of nitrate plus nitrite from processed meat to colon cancer, and nitrite to gastric cancer. The Panel recommended that further large-scale prospective studies be carried out on NDMA, nitrite and nitrate intake and risk of CRC and its subtypes, as well as on nitrite and gastric cancer subtypes. There is insufficient evidence for a positive association between nitrite alone in processed meat and other types of cancer (EFSA, 2017).

 

There is no clear evidence of carcinogenicity from nitrite per se in humans. However, a cancer risk may exist under conditions of endogenous nitrosation of ingested nitrate and/or nitrite (Health Canada, 2013).

 

 

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.]

 

Maekawa A, Ogiu T, Onodera H, Furuta K, Matsuoka C and Ohno Y, 1982. Carcinogenicity studies of sodium nitrite and sodium nitrate in F–344 rats. Food and Chemical Toxicology, 20, 25–33 [cited in EFSA, 2017.]

 

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