Calcium nitrite

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Reproductive Assessment by Continuous Breeding (RACB) study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 1988 - 10 April 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 RACB test protocol, and to GLP. The range of reproductive parameters was not as extensive as that indicated by current guidelines.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1990

Report date:

1990

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 Reproductive Assessment by Continuous Breeding (RACB) protocol

Version / remarks:

[See below for a brief description of the methods.]

Deviations:

no

Principles of method if other than guideline:

The RACB protocol consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include Task 1, dose finding: Task 2, continuous breeding phase: Task 3, identification of the affected sex: and Task 4, offspring assessment. Task 1 is conducted to determine suitable doses for the continuous breeding phase. The test chemical is administered for 14 consecutive days and the maximum tolerated dose (MTD) is estimated. Task 2 is designed to determine the effect of the estimated MTD and two lower dose levels on fertility and reproduction. In this phase, treatment is continued for 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter). If the fertility is significantly affected, Task 3 (a crossover mating trial) is conducted' to determine whether the male, female or both sexes are affected. Task 4 is designed to evaluate reproductive performance in the offspring (second generation) from the final Task 2 litters. If the fertility in the first generation animals (Task 2) is significantly affected, Task 4 is performed using second generation animals from the control and all three dose groups. If the overall response during Task 2 is negative, Task 4 is performed using second generation animals from the control and high dose groups only. At the conclusion of both Tasks 3 and 4, experimental animals are necropsied: the liver, kidneys, testes, epididymides, prostate, and seminal vesicles with coagulating glands are weighed and fixed for possible histopathologic evaluation. In addition, vaginal smears are prepared for 12 consecutive days prior to necropsy to check the effect on the estrous cycle. For male mice, sperm are studied in detail to evaluate the effect on sperm density, sperm motility, and sperm head morphology.

In the present study, Task 3 was not conducted because sodium nitrite treatment had no adverse effect on fertility and/or reproduction.

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:

mouse

Strain:

other: Swiss CD-1

Remarks:

Albino mice

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY for Task 1 and Portage, MI for Task 2 due to availability within the necessary time constraints)
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: (P) 11 wks for Task 2 (8 wks for Task 1 and 11 wks for Task 1 repeats); (F1) 3 wks for Task 4
- Weight at study initiation: (P) Males: 27.60-28.76 g (week 1); Females: 24.04-24.58 g (week 1); (F1) Males: 9.92-11.25 g (range of average pup weight at weaning); Females: 9.17-10.67 g (range of average pup weight at weaning)
- Fasting period before study: no data
- Housing: solid bottom polycarbonate cages with SaniChip bedding and stainless steel wire lids (2/cage for Task 1; bredding pairs or individually for Task 2)
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): rodent meal pellets (NIH-07 diet) ad libitum
- Water (e.g. ad libitum): distilled water ad libitum
- Acclimation period: 2 weeks (Tasks 1 and 2)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 19 May 1988 To: 10 April 1989

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared at least every 3 weeks by mixing sodium nitrite with deionized drinking water on a weight-to-volume basis. Each dose level was independently formulated.

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

Details on mating procedure:

- M/F ratio per cage: 1/1 (breeding pair)
- Length of cohabitation: 98 days
- Proof of pregnancy: vaginal plug (Task 4 only)
- Further matings after two unsuccessful attempts: not applicable
- After successful mating each pregnant female was caged (how): no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An aliquot of each formulation, the control, and the bulk chemical were sent to Research Triangle Institute for reference analysis during week 1 of Task 1, weeks 1, 6, 10, and 14 of Task 2 and weeks 20 and 28 for Task 4.

Duration of treatment / exposure:

105 days (P generation); not specified (F1 generation)

Frequency of treatment:

Continuously

Details on study schedule:

In Task 4 (offspring assessment), the last litter from Task 2 was reared, weaned, and kept to sexual maturity (74 ± 10 days) while housed by sex (maximum 2/cage) and receiving the same treatment as P0 animals. At sexual maturity, a male and female from different litters within the same treatment group were cohabited for 7 days and then housed singly until delivery.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.06 other: % (w/v)

Remarks:

Equivalent to approximately 131 mg/kg bw/day (P0 males) and 123 mg/kg bw/day (P0 females).

Doses / concentrations 2

Dose / conc.:

0.12 other: % (w/v)

Remarks:

Equivalent to approximately 273 mg/kg bw/day (P0 males) and 254 mg/kg bw/day (P0 females).

Doses / concentrations 3

Dose / conc.:

0.24 other: % (w/v)

Remarks:

Equivalent to approximately 437 mg/kg bw/day (P0 males) and 412 mg/kg bw/day (P0 females).

Doses / concentrations 4

Dose / conc.:

0.24 other: % (w/v)

Remarks:

Equivalent to approximately 433 mg/kg bw/day (P1 males) and 556 mg/kg bw/day (P1 females).

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for Task 2 were anicipated to be selected on the basis of a preliminary study (Task 1) so that the highest dose will be expected not to depress weight gain by more than 10% and will permit >90% survival. However, to avoid dehydration and methamoglobinemia, 0.24% was chosen as the high dose in Task 2. The middle dose is selected to produce little or no systemic toxicity, while the low dose is designed to be a "no-effect level".
- Rationale for animal assignment (if not random): All study animals were individually identified and assigned to treatment groups using a stratified randomisation procedure based on body weights.
- Other: The last litter born during the holding period following the continuous breeding phase (Task 2) is reared by the dam until weaning, after which treatment is initiated by the same route and at the same concentration as during Task 2. These animals are used for assessment of second generation fertility (Task 4).

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no data

BODY WEIGHT: Yes
- Time schedule for examinations: weeks 1 (precohabitation phase of Task 2), 2, 3, 6, 8 and 10

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weeks 1 (precohabitation phase of Task 2), 2, 6, 8 and 10

Oestrous cyclicity (parental animals):

Not conducted on P0 animals. For F1 animals, vaginal smears were prepared for 12 consecutive days prior to necropsy to check the effect on the estrous cycle.

Sperm parameters (parental animals):

Parameters examined in F1 male parental generation: testis weight, epididymis weight, sperm count in epididymides, sperm motility and sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no data

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, live births, proportion of pups born alive, pup body weight immediately after birth

GROSS EXAMINATION OF DEAD PUPS: no

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no data

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no data

Postmortem examinations (parental animals):

Not conducted on P0 animals.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were necropsied at the end of Task 4.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY: no data

HISTOPATHOLOGY / ORGAN WEIGHTS
Following the conclusion of Task 4, liver, kidneys, testes, epididymides, prostate, and seminal vesicles with coagulating glands were fixed for possible histopathologic evaluation. Organs weighed in F1 animals included liver, kidney (with adrenal glands attached), cauda epididymis, epididymis, prostate, seminal vesicles, testis and ovary. Kidney and liver from F1 animals (10/sex/group) were evaluated histologically.

Statistics:

For Task 2 data, the Cochran-Armitage test is used to test for a dose-related trend in fertility. The number of litters and the number of live pups per litter are computed on a per fertile pair basis and then treatment group means determined. The proportion of live pups is defined as the number of pups born alive divided by the total number of pups produced by each pair. The sex ratio is expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair. Dose group means for these parameters are tested for overall differences using the Kruskal-Wallis test and for ordered differences using Jonckheere's test. Pairwise comparisons of treatment group means are performed by applying the Wilcox-Mann-Whitney U test.

To remove the potential effect of the number of pups per litter on the average pup weight, an analysis of covariance is performed. The covariate used is average litter size, including live and dead pups. Least squares estimates of dose group means, adjusted for litter size, are computed and tested for overall equality using an F-test and pairwise equality using a t-test. To control for possible sex differences, these analyses are performed on males, females, and both sexes combined. An analysis of covariance is also used to adjust organ weights for total body weight. Unadjusted body and organ weights are analyzed using the Kruskal-Wallis and Wilcox-MannWhitney U tests. Dose-related trends are tested for by Jonckheere's test.

All body weight and feed consumption are analyzed in-house using nonparametric statistics. Group means, standard deviations, and standard errors are calculated for each dose level and for each sex. Jonckheere's test is performed to obtain evidence of any increasing or decreasing trends. Significance among dose groups is calculated using Shirley's test if trend is evident or Dunn's test if no trend is evident.

Reproductive indices:

Fertility (number producing a litter/number of breeding pairs), litters per pair, cumulative days to litter

Offspring viability indices:

No data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were reported.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

4, 1 and 2 animals died during the study (Task 2) in the low, mid and high dose groups, respectively. There were three mortalities in the control group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights for males in the treated groups were always within 10% of the corresponding control values. For females, body weights varied with gestation but were generally within 10% of the control values.

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 was significantly lower during weeks 1, 2, 6, and 14 in the 0.24% group and week 2 in the 0.12% group.

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

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All of the pairs were fertile in both control and treatment groups. 95% of the breeding pairs in the control and 0.12% groups and 100% of the pairs in the 0.06 and 0.24% groups delivered 4 litters each. [Data from pairs in which one or both partner(s) died during cohabitation were excluded.]

The reproductive performance of breeding pairs receiving sodium nitrite was similar to the control pairs with respect to all endpoints (average number of litters per pair, live pups per litter (male, female, or combined), proportion and sex of pups born alive, live pup weights (male, female, or combined), and adjusted live pup weights). The response was negative when the combined data for all litters were analysed as well as when values for each litter were analysed independently. The cumulative days to litter, dam weights at delivery, and dam weights at final litter were also not affected by sodium nitrite treatment.

Details on results (P0)

The NTP investigators considered that sodium nitrite treatment had no adverse effect on fertility and/or reproduction in Task 2.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive parameters

Effect level:

437 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No reproductive effects seen at highest tested dose

Remarks on result:

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

2

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive parameters

Effect level:

412 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No reproductive effects seen at highest tested dose

Remarks on result:

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

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights for treated F1 parental animals at weaning and during study weeks 28-30 were very similar to control values.

The group mean values for male terminal body weights were almost identical for control and treated animals. Female body weights were unaffected by sodium nitrite treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Water consumption values during the week of cohabitation were similar for control and treated animals.

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):

In males, there were also no significant differences with respect to liver, kidneys, epididymis, prostate, seminal vesicles, and right testis weights, though the absolute right cauda epididymal weights were significantly reduced by 9% in the 0.24% group; relative organ weights were similar for control and treated animals. Female absolute liver, kidneys, and ovary weights were not affected by sodium nitrite treatment; a small increase in relative kidney weight was apparent.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The incidence and severity of microscopic lesions in the kidney and liver were similar in control and treated animals.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no apparent effects on estrual cyclicity or the average estrous cycle length in treated F1 parental females.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In F1 parental males, epididymal sperm motility, sperm count, and percentage of abnormal sperm were unaffected by sodium nitrite treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating and fertility indices in the control and treated groups were essentially the same. The reproductive performance of second generation (F1) fertile pairs was also not affected for any of the endpoints (number of live F2 pups per litter, proportion of pups born alive, sex of pups born alive, live pup weights, and adjusted pup weights).

Details on results (P1)

The NTP investigators considered that there were no effects on fertility and/or reproduction in Task 4.

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

433 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No reproductive effects seen at highest tested dose

Remarks on result:

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

2

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

556 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No reproductive effects seen at highest tested dose

Remarks on result:

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

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were reported.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The proportion of F1 pups born alive in the final Task 2 litters and the postnatal survival to age 21 days were not affected by sodium nitrite consumption.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Live male and female pups weights were significantly lower in the 0.24% group on days 7, 14, and 21. The decreases in pup weights were dose-related but the differences were significant at the high dose only. Whether this represents a direct toxic effect of sodium nitrite or is secondary to maternal dehydration cannot be determined from these data.

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

F1 pup weights in the final litter were reduced on days 7, 14, and 21 in 0.24% group. The NTP investigators could not determine whether this is due to a direct toxic effect of sodium nitrite on pup development, or is secondary to reduced maternal fluid consumption during lactation from these data. However, it was suggested that higher doses would have more severely compromised pup growth or survival, supporting the use of the 0.24% level as the MTD for this study.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

437 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No reproductive or developmental effects seen at highest tested dose

Remarks on result:

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

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

412 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No reproductive or developmental effects seen at highest tested dose

Remarks on result:

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

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

The NTP investigators concluded that, in the absence of effects on reproductive and fertility indices, sodium nitrite is not a reproductive toxicant in this strain of mouse at the dose levels tested.

The EFSA Panel reported that no reproductive and developmental effects were observed in the study (up to respective doses of 437 and 412 mg/kg bw/day for males and females). The Panel considered the effects on pup weight in the F1 generation animals secondary to the decreased maternal water consumption but noted that at higher doses an effect cannot be excluded.

Applicant's summary and conclusion

Conclusions:

No effect on fertility or any of the other assessed reproductive parameters was seen in an NTP continuous breeding study involving the exposure of mice to drinking water containing sodium nitrite at up to 0.24% (w/v, equivalent to approximate dose levels of 437 and 412 mg/kg bw/day for males and females, respectively) for 105 days

Executive summary:

In an oral reproductive toxicity study, conducted according to the NTP RACB test protocol and to GLP, sodium nitrite was provided in the drinking water to Swiss CD-1 mice (20/sex/group) at 0.06, 0.12 or 0.24% (w/v; equivalent to approximate dose levels of 131, 273 or 437 mg/kg bw/day for males and 123, 254 or 412 mg/kg bw/day for females) for 105 days. The study encompassed a 7-day pre-cohabitation period followed by a 98-day cohabitation period. Control animals (40/sex) received vehicle only. The dose levels for the main test (Task 2) were established on the basis of a range-finding study (Task 1). Clinical signs, growth and water consumption, as well as a range of fertility/reproductive parameters (including litters/pair, live pups/litter and pup body weights at birth) were monitored in Task 2.

 

In Task 4 (offspring assessment), litters from Task 2 was reared, weaned, and kept to sexual maturity (9-12 weeks) while receiving sodium nitrite via drinking water at 0.24% (w/v; equivalent to approximate dose levels of 433 and 556 mg/kg bw/day for males and females, respectively). Fertility parameters evaluated were similar to Task 2. All F1 animals were necropsied at the end of Task 4, whereby select organs were weighed and a variety of sperm parameters were measured; estrous cyclicity was also monitored for 12 days prior to necropsy. The kidney and liver were evaluated microscopically.

 

In Task 2, body weights were not significantly reduced by treatment though water consumption was generally lower in the high dose group. Mortality was observed in both control and treated groups, showing no apparent trend. There were no significant effects on fertility and reproductive performance was also unaffected by treatment. At the highest tested dose level, F1 pup body weights were reduced on postnatal days 7, 14, and 21, though these had normalised by the end of the study. No effects on organ weights were observed in necropsied animals (Task 4), aside from a reduction in absolute cauda epidydimal weight (by 9% compared to controls) in males and a slight increase in relative kidney weight in females. Epidydimal sperm motility, sperm count, and percentage of abnormal sperm were also not affected by sodium nitrite treatment and there were no apparent effects on estrual cyclicity or the average estrous cycle length in treated females. Histopathological findings in liver and kidney were similar between control and treated animals. The reproductive performance of second generation (F1) fertile pairs was unaffected by treatment (NTP, 1990). [Task 3 (cross-over mating trial) was not conducted because sodium nitrite treatment had no adverse effect on fertility and/or reproduction and, as such, there was no need to determine the affected sex.]

 

The EFSA Panel also did not consider the decrease in cauda epidydimal weight to be evidence of reproductive toxicity and deemed the effects on F1 pup weight secondary to the decreased maternal water consumption. Respective NOAELs of 437 and 412 mg/kg bw/day were established for first-generation parental males and females, with analogous levels of 433 and 556 mg/kg bw/day for second-generation parental males and females, respectively, covering both reproductive and developmental parameters (EFSA, 2017).