Calcium nitrite

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

 

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 (NTP, 1990).

Link to relevant study records

Reference

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.

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

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

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.

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).

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).

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).

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

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

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: 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.

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

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.

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.

Critical effects observed:

not specified

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: 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).

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

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.

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.

Critical effects observed:

not specified

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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.

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.

Critical effects observed:

not specified

Reproductive effects observed:

no

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.

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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

412 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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

 

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 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 evidently 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).

 

In the 14-week oral toxicity study in rats, reduced sperm motility was observed at 115 and 310 mg/kg bw/day; females displayed no effects on vaginal cytology parameters at up to 345 mg/kg bw/day (NTP, 2001).

 

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”, nor presumably overt fertility/reproductive effects, 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.]

 

No studies in humans that directly evaluated the potential for prenatal exposure to sodium nitrite to

cause adverse effects on fetal viability, growth, morphology or functional parameters were available (OECD, 2005).

 

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.]

 

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

Effects on developmental toxicity

Description of key information

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

 

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 (Globus and Samuel, 1978).

 

In addition, no convincing evidence of developmental toxicity was seen in the offspring of mice exposed to sodium nitrite via drinking water 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 in an NTP continuous breeding study (NTP, 1990). On this basis, an EFSA Panel established respective NOAELs of 437 and 412 mg/kg bw/day for F1 males and females, respectively (EFSA, 2017).

Link to relevant study records

Reference

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").

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

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

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

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

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

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

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

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.

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.

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.

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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

 

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 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 (Globus and Samuel, 1978).

 

No adverse developmental effects were seen up to postnatal day 140, following the gavage of pregnant rats with sodium nitrite at 80 mg/kg bw on GD 15 (Khera, 1982).

 

In addition, in an NTP RACB study in mice, 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 (NTP, 1990). The EFSA Panel deemed the effects on F1 pup weight secondary to the decreased maternal water consumption. On this basis, an EFSA Panel established respective NOAELs of 437 and 412 mg/kg bw/day for F1 males and females, respectively (EFSA, 2017).

 

Further, 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”, nor presumably overt fertility/reproductive effects, 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.]

 

Developmental toxicity was tested in mice, rats and hamsters at doses up to 23, 10 and 23 mg

sodium nitrite/kg bw/day (FDA, 1972a) and up to 32, 10 and 32 mg potassium nitrite/kg bw/day (FDA, 1972b). Only a slight effect (skeletal retardation) was observed at the high dose in rats

treated with sodium and potassium nitrite [this effect was evidently not considered to be of concern by the Panel in the derivation of an ADI for nitrite] (EFSA, 2017).

 

No teratogenic effects were observed in tests with nitrite (IARC, 2010).

 

 

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.]

 

FDA (1972a). Food and Drug Administration. Teratologic evaluation of FDA 71-9 (sodium nitrite). Report nr. FDABF-GRAS-061. Maspeth, NY: Food and Drug Research Laboratories, Inc [cited in EFSA, 2017.]

 

FDA (1972b). Food and Drug Administration. Teratologic evaluation of FDA 71-10 (potassium nitrite). Report nr. FDABF-GRAS-065. Maspeth, NY: Food and Drug Research Laboratories, Inc [cited in EFSA, 2017.]

 

Khera (1982). Reduction of teratogenic effects of ethylenethiourea in rats by interaction with sodium nitrite in vivo. Food and Chemical Toxicology 20, 273-278 [cited in NTP, 1990; OECD, 2005.]

 

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

Toxicity to reproduction: other studies

Description of key information

No data identified.

Additional information

No data identified.

Mode of Action Analysis / Human Relevance Framework

No data identified.

Justification for classification or non-classification

No evidence of fertility, reproductive or developmental effects was observed in reliable studies with sodium nitrite. As such, classification of calcium nitrite for reproductive toxicity is not required, according to EU CLP criteria (EC 1272/2008).