Calcium nitrite

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Generally well-conducted study, albeit with only a single (low) dose level. The OECD considered this study to be reliable 2 ("valid with restrictions").

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Effect of maternally administered sodium nitrite on hepatic erythropoiesis in fetal CD-1 mice.

Author:

Globus M and Samuel D

Year:

1978

Bibliographic source:

Teratology 18, 367-378

Reference 2

Reference Type:

review article or handbook

Title:

SIDS Initial Assessment Report (SIAR) for SIAM 20. Sodium nitrite, CAS No: 7632-00-0.

Author:

OECD

Year:

2005

Bibliographic source:

Organization for Economic Co-operation and Development. Screening Information Dataset.

Report date:

2005

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The study was initiated to investigate the possible embryotoxic effects of (sodium) nitrite ingested by mammals during pregnancy, and in particular, the influence on fetal haematopoiesis.
- Short description of test conditions: Female mice were gavaged with sodium nitrite for 14-18 days during pregnancy before sacrifice and examination. Embryonic liver was isolated for analysis.
- Parameters analysed / observed: Classical parameters of embryotoxicity, such as the number of offspring per litter, weight per embryo, number of resorption sites per litter and fetal mortality were examined along with haemopoietic cell counts and skeletal abnormalities.

GLP compliance:

not specified

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:

not specified

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Montreal
- Age at study initiation: 8-10 weeks (females)
- Weight at study initiation: 22-28 g (females)
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): standard rat cubes supplied by Maple Leaf Mills Ltd., Ontario
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: no data

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

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: no data

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

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Males (four- to five-month-old) were mated with females when the latter were found to be in the estrous phase, on the basis of vaginal smears.
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
- Any other deviations from standard protocol: not applicable

Duration of treatment / exposure:

14, 16 or 18 days (during gestation)

Frequency of treatment:

Daily

Duration of test:

Until gestation day (GD) 19

Doses / concentrations

Dose / conc.:

0.5 other: mg/day

Remarks:

Equivalent to about 17.9-22.7 mg/kg bw/day.

No. of animals per sex per dose:

42 treated females in total

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the maternal dose of sodium nitrite was intentionally kept as low as possible
- Rationale for animal assignment (if not random): no data

Examinations

Maternal examinations:

Maternal examinations were limited to peripheral blood counts.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: no data

Fetal examinations:

- External examinations: Yes: The embryos were examined for gross anatomical defects.
- Soft tissue examinations: Yes: No data
- Skeletal examinations: Yes: The axial and appendicular skeletons of 37 control and 42 treated embryos were examined.
- Head examinations: No data

Statistics:

The haemopoietic cell count data, expressed as mean ± standard deviation, were compared to determine whether differences between means were statistically significant. A student's T-test was used for this analysis and differences with P < 0.05 were considered significant.

Indices:

A number of classical parameters of embryotoxicity (number of offspring per litter, weight per embryo, number of resorption sites per litter and fetal mortality (expressed as percent of total offspring)) were examined.

Historical control data:

Not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

not examined

Description (incidence and severity):

[Presumably overt effects were not seen]

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Description (incidence):

[Presumably overt effects were not seen]

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no significant differences between mean maternal peripheral blood counts of treated and control mice. Also, when erythroid cells from corresponding stages of gestation were compared, no morphological differences were detected between controls and their nitrite-treated counterparts at the light microscope level.

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:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not examined

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no significant differences in the number of resorption sites per litter between the control and treated groups.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no significant differences in fetal mortality (expressed as percent of total offspring) between the control and treated groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No effects on fetal mortality or resorptions at the only tested dose level

Remarks on result:

other: The OECD reported the NOAEL as 20 mg/kg bw/day.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in weight per embryo between the control and treated groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no significant differences in fetal mortality or in the number of offpsring per litter between the control and treated groups.

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no significant differences in the number of offspring per litter between the control and treated groups.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No gross anatomical defects were reported.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Both control and treatment groups displayed skeletal abnormalities, though the frequency of their occurrence appeared to be independent of treatment. Sternal abnormalities (including asymmetrical fusion, inhibited ossification and bilateral ossification of sternabrae) were evident in 43.12% of control embryos and 47.6% of treated embryos. Both the incidence and severity of asymmetrical fusion appeared to increase in the treated group, but overall, the increase in skeletal abnormalities was not statistically significant.

There appeared to be a tendency toward talipomanus and talipes in the treated group.

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At GD 14 there was a significant increase (p < 0.01) in polychromatophilic erythroblasts, accompanied by a relative decrease in pro- and basophilic erythroblasts. Orthochromatophilic erythroblasts were significantly increased and mature erythrocytes significantly reduced (both p < 0.05).

At GD 16, the tendency for early precursor cells to differentiate at an increased rate was manifested in a highly significant increase (p < 0.01) in orthochromatophilic erythroblasts and mature erythrocytes. The latter increase was accompanied by an apparent decrease in the relative number of precursor cells and polychromatophilic erythroblasts.

At GD 18, the proportion of mature erythrocytes in the treated group was not statistically significantly different from the control value.

There were no significant differences between mean fetal peripheral blood counts of treated and control mice. Also, when erythroid cells of corresponding stages of maturation were compared, no morphological differences were detected between controls and their nitrite-treated counterparts at the light microscope level.

Overall, there did not appear to be a considerable change in fetal white cell populations and no morphological differences were detected between white cells of corresponding stages of maturation.

Details on embryotoxic / teratogenic effects:

Fetal mortality, resorptions, the mean number of offspring per litter, the mean weight per embryo and the incidence of skeletal malformations, were not significantly different from controls.

In the earliest stages of hepatic erythropoiesis that were examined (GD 14), immature forms constituted the bulk of the erythroid cells, and there was a subsequent rapid increase in more mature cells. However, sodium nitrite treatment resulted in a significant increase in the embryonic hepatic production of erythroid cells, when compared with controls. This apparent stimulation of erythropoiesis was reflected in an increased proportion of nucleated polychromatophilic erythroblasts in the hepatic erythroid cell population at GD 14 and a subsequent increase over control, in the proportion of mature erythrocytes at GD 16. This increase in mature RBCs was not apparently sustained at GD 18. The investigators considered that this result was somewhat unexpected, but may be explained by the fact that hepatic erythropoiesis reaches its definitive phase at GD 18-19 in the mouse, whereby the liver is supplanted by the bone marrow and spleen as the major sites of erythropoiesis.

The investigators considered that the observed stimulation of fetal erythropoiesis was likely caused by maternally administered sodium nitrite traversing the placenta and inducing methaemoglobinaemia.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on external/skeletal abnormalities, fetal mortality, weight per embryo or in the number of offpsring per litter at the only tested dose level

Remarks on result:

other: The OECD reported the NOAEL as 20 mg/kg bw/day.

Remarks:

The OECD noted that although there was a treatment-related increase in embryonic hepatic production of erythroid cells at GD 14-16, this effect was not sustained at GD 18, likely due to a normal developmental shift in the main site of erythropoiesis (liver to the bone marrow and spleen); as no increase in red blood cell counts was observed in the peripheral circulation, the functional significance of these findings remains unclear.

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Gavage treatment of pregnant mice with sodium nitrite at approximately 20 mg/kg bw/day from gestation day (GD) 1 to GD 14, 16 or 18 produced no convincing evidence of developmental toxicity.

Executive summary:

In a reliable (non-guideline) study, sodium nitrite was administered to 42 pregnant CD-1 mice by oral gavage at 0.5 mg/day (approximately 20 mg/kg bw/day) from gestation day (GD) 1 until sacrifice on GD 14, 16 or 18. Control animals (n=37) received vehicle only. Parameters evaluated included litter size, resorption sites, fetal mortality and fetal weight as well as gross anatomical defects and skeletal abnormalities. No developmental effects were reported. In addition, the embryonic liver was isolated for analysis of haemopoietic cell counts (as the liver is the main site of erythrocyte production on GD 12-19 in the mouse).

A treatment-related increase in embryonic hepatic production of erythroid cells at GD 14-16, this effect was not sustained at GD 18, likely due to a normal developmental shift in the main site of erythropoiesis (liver to the bone marrow and spleen); as no increase in red blood cell counts was observed in the peripheral circulation, the functional significance of these findings remains unclear.