Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reliable study with sodium nitrate itself is present. In a reliable OECD screening study in rats with potassium nitrate no effects were found up to the highest dose tested (1500 mg/kg bw/d). This is supported by the less reliable studies with sodium nitrate itself. In a 2-generation study in dogs doses with up to 1000 mg sodium nitrate/L in the drinking water no indication of significant adverse effects on development, fertilization or hypothyroidism was observed. An expert statement was written and together with the available data showing no effects, an additional study is not considered necessary. The overall conclusion for sodium nitrate is that there is no evidence that the substance may present a risk to fertility.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 7, 2002 – October 14, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed with a substance analogue and the data are read across.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: males were 64 days of age on day of arrival; females were 61 days of age on day of arrival
- Weight at study initiation: 225 – 325 grams for male rats; 161 – 219 for female rats
- Fasting period before study:
- Housing: Animals were individually housed except for during the cohabitation and lactation period in wire mesh suspended stainless steel cages which conformed with GLP requirements, During cohabitation each pair of rats were housed in the male rat’s cage. Beginning no later than Day 20 of gestation, female rats were individually housed in polyethylene shoebox cages containing nesting material with wire mesh lids. Each dam and litter was housed in a common nesting box during the lactation/postnatal period.
- Diet (e.g. ad libitum): Purina Certified Rodent Diet #5002; as libitum
- Water (e.g. ad libitum): automatic dispenser; ad libitum and when females and litters were housed in shoebox cages via water bottle; ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 22
- Humidity (%): 43 – 66
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs

IN-LIFE DATES: Jan 8, 2002 – Feb 23, 2002
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on exposure:
Dose calculations: Individual doses were calculated based on the most recent weekly body weights and were adjusted each week to maintain the targeted dose level for all rats in the General Toxicity groups (i.e., mg/kg/day). For female rats in te Reproduction groups, individual doses were calculated based on the most recent body weights and were adjusted to maintain the targeted dose level (i.e., mg/kg/day). All doses were administered by volume of 10 mL/kg after correcting for concentration of the test mixture. Control animals received the vehicle only at the same volume as the test groups.

Dose preparations: The test substance (011101-3D) was ground in a Krups coffee mill (Model 203) prior to use and again upon receipt of additional test substance (020122-1D). A quantity sufficient to cover the grinding blade was added to the coffee mill and ground to a fine powder. Appropriate amounts of ground test material were accurately weighed into a 100 mL volumetric flask and diluted to volume with distilled water for each of the low, mid and high concentrations. Given that there was visual evidence (i.e. settling of test substance to bottom of cup) of a small amount of precipitate , the dosing mixtures were constantly stirred on a magnetic stir plate while being sampled to dose the test animals during the study.

Dose frequency: Each animal was dosed by oral intubation using a stainless steel balltipped gavage needle attached to an appropriate syringe. Dose administration was daily (7 days/week) for all adult animals as follows:
Male rats: Reproduction/General toxicity groups: during two-week premating and two-week mating periods for at least 28 days of exposure.
Female rats: Reproduction groups: during two-week premating, two-week mating, gestational and lactational periods. General Toxicity groups: for at least 28 days of exposure.
Details on mating procedure:
- Length of cohabitation: 14 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance was assumed to be homogenous and stable at the time drawn by syringe to dose the test animals. Analysis of dosing mixtures, therefore, were limited to concentration verification of representative preparations intended for the control, low, intermediate and high dose levels in the study. Representative dosing mixtures of each concentration during the study were provided to the analytical department at three time points during the study (prior to animal exposure, near the middle (Test days 24 and 28) and near the end of the study (Test day 45). Vehicle control samples were inadvertently not submitted for analysis. Each dose preparation was evaluated by flame atomic absorption spectroscopy for total potassium (SOAC Official Method 975.03)(1988). A reference standard of potassium (999 ug/ml) , supplied by EM Science, was used for calibration.
Duration of treatment / exposure:
Animals on the study were divided between two subgroups (toxicity and reproductive subgroups). The exposure period for males and females in the toxicity subgroup was 28 days. The exposure period for reproductive subgroup males was at most 28 days. The exposure period for reproductive subgroup females was at most 53 days (14 days pre-mating, 14 days mating, and gestational and lactational periods up to lactation day 4).
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 250, 750, and 1,500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on parameters assessed in a range-finding study at concentrations up to 1,000 mg/kg/day.
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for viability and cage-side observations were performed daily during acclimation, premating and mating, gestation, and lactation periods, except when scheduled detailed observations were conducted. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were performed and recorded at least once during the acclimation period for all male and female rats. Observations were performed and recorded approximately once per week during the premating and mating periods for females of the reproduction groups during the gestational days (GD7, GD14 and GD20) and lactational (LD4 only) periods. Female rats were evaluated for adverse clinical signs during parturition. Maternal behavior was checked on LD0 and LD4 and recorded. The date and clock time of all observations and/or mortality checks was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at least twice during the acclimation period (including the day after receipt) before pairing and mating. All male rats were weighed weekly during the premating and mating periods and at the time of sacrifice. Mated females were weighted on GD0, 7, 14 and 20, and on the day of delivery (LD0) and LD4 (prior to terminal sacrifice). Females showing no evidence of mating were assigned a GD0 after cessation of cohabitation and body weights were measured accordingly. Females in the General Toxicity Groups were weighed weekly and at the time of sacrifice. Body weight gains were calculated for males and females during each appropriate interval.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Although not a feeding study, food consumption was determined weekly during the premating period (no mating period) for all males and females. Individual food consumption was measured and recorded weekly thereafter for the females in the general toxicity groups and during the gestational period for the females in the reproductive groups. Food consumption was also recorded on LD0 and LD4. The data were then used to calculate food efficiency for the associated intervals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Data from food consumption were used to determine food efficiency for associated intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: See detailed clinical observations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of treatment
- Anaesthetic used for blood collection: Yes. Isoflourane anesthesia ) collected via orbital sinus bleeding.
- Animals fasted: Yes, 18 hours prior to blood collection
- How many animals: 5 males and 5 females/dose level
- Parameters examined: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28 of treatment
- Animals fasted: Yes. 18 hours prior to blood collection
- How many animals: 5 males and 5 females/dose level
- Parameters examined: calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alanine aminotransferase (SGPT), serum aspartate aminotransferase (SGOT), gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin, total serum protein, globulin, total cholesterol, alkaline phosphatase and magnesium measurements.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the final days of treatment
- Dose groups that were examined: Five male and five females/dose group (including controls).
- Battery of functions tested: sensory activity / grip strength / motor activity: excitability, autonomic function, gait and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behavior) and other abnormal clinical signs including but not limited to convulsions, tremors, unusual or bizarre behavior, emaciation, dehydration, and general appearance. The rats were observed in random without the observer aware of the dose group.

Motor activity was also evaluated. Each animal was evaluated for a single one-hour phase, with photobeam counts accumulated over six, 10-minute intervals. Total movements (consisting of fine and active movements) was considered an appropriate measure for the assessment of potential behavior effects in this screening level study.)
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, survival indices, presence of gross anomalies, weight gain
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- Organs: Necropsy: Gross necropsy of all males and females included an initial examination of external surfaces and orifices, as well as the cranial, thoracic and abdominal cavities and their contents. Rats were examined for gross lesions. Gross lesions were retained in neutral buffered 10% formalin (NBF).
- Reproductive groups: Special attentionwas paid to the organs of the reporductive system. For male rats, the testes and epididymides, seminal vesicles with coagulating gland and prostate were retained. The testes and epididymides were weighed. The testes were fixed in Bouin's solution for 4-12 hours (depending on size) before being retained in 70% alcohol. For females, special care was taken to examine the uterus for the presence of conceptuses. For each female, the ovaries were weighed; the ovaries, uterus and accompanying structures, and a mammary gland were retained in NBF. Subsequently, the fixed uterus from all adult female rats (including those that did not deliver litters and those that delivered pups) were stained with potassium ferricyanide to confirm pregnancy status and to determine post-implantation loss.
Rats were evaluated for gross lesions. Pregnancy status and uterine contents of female rats were recorded. Aborted fetuses and/or delivered pups were examined to the extent possible. Organs and tissues were excised, weighed, preserved and/or stained and implantation sites counted as described for those animals sacrificed by design.
- General Toxicology Groups: At scheduled sacrifice, all survivors were euthanized by exsanguination from the abdominal aorta under isoflourane anesthesia. All animals were subjected to a full necropsy that included examination of the external surface of the body, all orifices and the thoracic, abdominal and cranial cavities and their contents. The liver, kidneys, adrenals, brain, heart, thymus, spleen, ovaries, testes and epididymides (of all animals sacrificed by design) were weighed wet as soon as possible after dissection to avoid drying. The following organs and tissues from all animals were preserved in NBF for possible future histopathological examination: all gross lesions, lungs, brain- including sections of the medulla/pons, cerebellar cortex and cerebral cortex, spinal cord (3 levels: cervical, mid-thoracic, and lumbar), eyes, pituitary, thyroid/parathyroid, thymus, trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys, adrenals, pancreas, ovaries, testes, uterus (with attached urinary bladder, cervix and vagina), accessory sex organs (epididymides, prostate, and seminal vesicles), female mammary gland, skin, aorta, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, representative lymph node, and peripheral nerve (sciatic).

HISTOPATHOLOGY: Yes
-Organs: Histopathologic examination was performed on the preserved organs and tissues of the Reproductive and General Toxicity Group animals from the control (Groups I and II) and high dose (Groups VII and VIII). In addition, gross lesions of potential toxicological significance noted in any test groups were also examined. Microscopic findings were graded.

Statistics:
Mean and standard deviations were calculated for all quantitative data. Except for clinical pathology data were the contract laboratory, Huntingdon Life Sciences, elected to use statistics to aid in the data interpretation; no further statistical treatment of the study was conducted due to small group sizes.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY: were no treatment-related deaths and no signs of overt clinical toxicity.

BODY WEIGHT AND WEIGHT GAIN: There were no effects on body weight, food consumption, or food efficiency.

WATER CONSUMPTION AND COMPOUND INTAKE: There were no effects of test-substance treatment on food consumption in males. There were no effects of food consumption on females during pre-mating; during weeks 3, 4 and 5 for females in the General Toxicity Group; or during gestation and lactation. Food consumption was not measured during the mating period. Food efficiency was also unaffected by treatment.

HAEMATOLOGY:
No test substance related haematological changes were associated with the test substance treatment.

CLINICAL CHEMISTRY A slight increase in blood urea nitrogen was observed in male and female rats at 1,500 mg/kg/day and in female rats at 750 mg/kg/day. Although outside the range of the historical control, the absence of other indicators of renal dysfunction (e.g., creatinine) discounted the clinical significance of this endpoint. Minimal changes in electrolyte levels in male rats (e.g., 10% decrease in potassium, 4% decrease in calcium, and 22% increase in phosphorus) and female rats (3% decrease in chloride, 4% decrease in magnesium) were observed at 1,500 mg/kg/day. These changes were within the range of historical control and were not considered to be of biological significance.

NEUROBEHAVIOUR: Functional observational battery (FOB) and motor activity tests identified no treatment-related changes in behavior, function, or motor activity.

ORGAN WEIGHTS: Mean organ weights and organ-to-body weight ratios for both the Reproduction and General Toxicity test groups, in general, were considered comparable to their respective control groups. Any slight increases or decreases from the control were incidental, not dose-related and judged not to be of toxicological importance.

GROSS PATHOLOGY: There were a number of gross observations correlated to microscopic findings. The dilatation of the uterus (horns) observed in several female rats from the General Toxicology Group was considered to be a function of the estrus stage (generally proestrus, but sometimes early estrus). These gross observations and others, along with their microscopic correlates, were all considered incidental background findings not attributable to administration of the test substance.

HISTOPATHOLOGY: No treatment-related histopathological changes were reported.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse (fertility or reproductive) effects at highest dose tested
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
There were no treatment-related deaths and no signs of overt clinical toxicity. There were no effects on body weight, food consumption, or food efficiency. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring.
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified

NOAEL:1,500 mg/kg/day (general toxicity)

NOAEL:1,500 mg/kg/day (reproduction/developmental toxicity)

LOAEL: No adverse effects were seen on general toxicity endpoints. No adverse effects were seen on reproduction/developmental toxicity endpoints

There were no treatment-related deaths and no signs of overt clinical toxicity. There were no effects on body weight, food consumption, or food efficiency. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring.

 

Conclusions:
Based on the results of a combined repeated dose toxicity study with a reproduction/ developmental toxicity screening performed according to OECD 422 guideline and GLP principles, the NOAEL of potassium nitrate was found to be >= 1,500 mg/kg/day for reproduction toxicity.
Executive summary:

A combined repeated dose toxicity study with a reproduction/ developmental toxicity screening performed according to OECD 422 guideline and GLP principles was performed with potassium nitrate. Male and female rats were exposed to 0, 250, 750 or 1500 mg/kg bw/ day. There were no treatment-related deaths and no signs of overt clinical toxicity. There were no effects on body weight, food consumption, or food efficiency. Mating performance and fertility were unaffected by the treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ratio, offspring bodyweight, or macropathology for offspring. Based on these data, the NOAEL of potassium nitrate was found to be >=1,500 mg/kg/day for reproduction toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. However, since the study was performed with a substance analogue and the data are read across, the Klimisch score is 2.
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 reliable study with sodium nitrate is present. A reliable 28-day oral OECD 422 study has been performed in rats (5 rats/sex/dose) via gavage, containing 50, 750 or 1500 mg/kg bw/day potassium nitrate. There were no treatment-related deaths and no signs of overt clinical toxicity. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring. Therefore, it was concluded that the NOAEL is >=1500 mg/kg bw/day (or higher, highest dose tested).

Based on an expert statement, no further studies need to be conducted. No fertility effects or effects on reproductive organs were observed up to and including the highest dose tested of 1500 mg/kg bw/d in an oral OECD 422 study in rats with potassium nitrate.

In a 2-generation study in dogs doses with up to 1000 mg sodium nitrate/L in the drinking water no indication of significant adverse effects on development, fertilization or hypothyroidism was observed. Sodium is an essential element for humans and the acceptable daily intake for sodium is 2.4 g/day (Dutch Voedingscentrum). In relation to this value exposure due to use as described under REACH is considered minimal.

Moreover, sodium nitrate has been used in fertilizers for over decades without an identifiable relation to reproductive or developmental effects.

JECFA and the former SCF derived an ADI of 3.7 mg/kg bw/d for nitrate, which was left unchanged by EFSA (EFSA, Nitrate in vegetables. Scientific opinion of the panel on contaminants in the food chain, 2008). This ADI was based on a 2-y study in rats with a NOEL of 500 mg sodium nitrate/kg bw/ (= 370 mg/kg bw/d nitrate)and a subchronic study with sodium nitrate in dogs with a NOEL of 370 mg/kg bw/d for nitrate. The OECD study with potassium nitrate in this dossier has a NOAEL of 1500 mg/kg bw/d which is equivalent to 62/101.1 x 1500 = 920 mg nitrate/kg bw/d, which is higher than the study on which the ADI was based. As the exposure assessment using this value indicates safe use, it is also considered safe for reproductive and developmental toxicity.

EFSA recently (June 2017) re-evaluated sodium nitrate and potassium nitrate as food additives. The Panel concluded that currently there was insufficient evidence to withdraw the ADI of 3.7 mg/kg bw/d for nitrate.

Justification for selection of Effect on fertility via oral route:

One study on the read-across substance potassium nitrate is available.

Effects on developmental toxicity

Description of key information

No reliable study with sodium nitrate is avaialble. In a reliable OECD screening study in rats with potassium nitrate no effects were found up to the highest dose tested (1500 mg/kg bw/d). In a 2-generation study in dogs doses with up to 1000 mg sodium nitrate/L in the drinking water no indication of significant adverse effects on development, fertilization or hypothyroidism was observed.

An expert statement was written and together with the available data showing no effects, an additional study is not considered necessary.

The overall conclusion for sodium nitrate is that there is no evidence that the substance may present a risk for developmental toxicity.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 7, 2002 – October 14, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed with a substance analogue and the data are read across.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
not specified
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: males were 64 days of age on day of arrival; females were 61 days of age on day of arrival
- Weight at study initiation: 225 – 325 grams for male rats; 161 – 219 for female rats
- Fasting period before study:
- Housing: Animals were individually housed except for during the cohabitation and lactation period in wire mesh suspended stainless steel cages which conformed with GLP requirements, During cohabitation each pair of rats were housed in the male rat’s cage. Beginning no later than Day 20 of gestation, female rats were individually housed in polyethylene shoebox cages containing nesting material with wire mesh lids. Each dam and litter was housed in a common nesting box during the lactation/postnatal period.
- Diet (e.g. ad libitum): Purina Certified Rodent Diet #5002; as libitum
- Water (e.g. ad libitum): automatic dispenser; ad libitum and when females and litters were housed in shoebox cages via water bottle; ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 22
- Humidity (%): 43 – 66
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs

IN-LIFE DATES: Jan 8, 2002 – Feb 23, 2002
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on exposure:
Dose calculations: Individual doses were calculated based on the most recent weekly body weights and were adjusted each week to maintain the targeted dose level for all rats in the General Toxicity groups (i.e., mg/kg/day). For female rats in te Reproduction groups, individual doses were calculated based on the most recent body weights and were adjusted to maintain the targeted dose level (i.e., mg/kg/day). All doses were administered by volume of 10 mL/kg after correcting for concentration of the test mixture. Control animals received the vehicle only at the same volume as the test groups.

Dose preparations: The test substance (011101-3D) was ground in a Krups coffee mill (Model 203) prior to use and again upon receipt of additional test substance (020122-1D). A quantity sufficient to cover the grinding blade was added to the coffee mill and ground to a fine powder. Appropriate amounts of ground test material were accurately weighed into a 100 mL volumetric flask and diluted to volume with distilled water for each of the low, mid and high concentrations. Given that there was visual evidence (i.e. settling of test substance to bottom of cup) of a small amount of precipitate , the dosing mixtures were constantly stirred on a magnetic stir plate while being sampled to dose the test animals during the study.

Dose frequency: Each animal was dosed by oral intubation using a stainless steel balltipped gavage needle attached to an appropriate syringe. Dose administration was daily (7 days/week) for all adult animals as follows:
Male rats: Reproduction/General toxicity groups: during two-week premating and two-week mating periods for at least 28 days of exposure.
Female rats: Reproduction groups: during two-week premating, two-week mating, gestational and lactational periods. General Toxicity groups: for at least 28 days of exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance was assumed to be homogenous and stable at the time drawn by syringe to dose the test animals. Analysis of dosing mixtures, therefore, were limited to concentration verification of representative preparations intended for the control, low, intermediate and high dose levels in the study. Representative dosing mixtures of each concentration during the study were provided to the analytical department at three time points during the study (prior to animal exposure, near the middle (Test days 24 and 28) and near the end of the study (Test day 45). Vehicle control samples were inadvertently not submitted for analysis. Each dose preparation was evaluated by flame atomic absorption spectroscopy for total potassium (SOAC Official Method 975.03)(1988). A reference standard of potassium (999 ug/ml) , supplied by EM Science, was used for calibration.

Details on mating procedure:
- Length of cohabitation: 14 days
Duration of treatment / exposure:
Animals on the study were divided between two subgroups (toxicity and reproductive subgroups). The exposure period for males and females in the toxicity subgroup was 28 days. The exposure period for reproductive subgroup males was at most 28 days. The exposure period for reproductive subgroup females was at most 53 days (14 days pre-mating, 14 days mating, and gestational and lactational periods up to lactation day 4).
Frequency of treatment:
daily
Duration of test:
tox group: 28 days
repro group: 53 days
Remarks:
Doses / Concentrations:
0, 250, 750, and 1,500 mg/kg/day (Doses were selected based on parameters assessed in a range-finding study at concentrations up to 1,000 mg/kg/day)
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for viability and cage-side observations were performed daily during acclimation, premating and mating, gestation, and lactation periods, except when scheduled detailed observations were conducted. All observations were recorded.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were performed and recorded at least once during the acclimation period for all male and female rats. Observations were performed and recorded approximately once per week during the premating and mating periods for females of the reproduction groups during the gestational days (GD7, GD14 and GD20) and lactational (LD4 only) periods. Female rats were evaluated for adverse clinical signs during parturition. Maternal behavior was checked on LD0 and LD4 and recorded. The date and clock time of all observations and/or mortality checks was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at least twice during the acclimation period (including the day after receipt) before pairing and mating. All male rats were weighed weekly during the premating and mating periods and at the time of sacrifice. Mated females were weighted on GD0, 7, 14 and 20, and on the day of delivery (LD0) and LD4 (prior to terminal sacrifice). Females showing no evidence of mating were assigned a GD0 after cessation of cohabitation and body weights were measured accordingly. Females in the General Toxicity Groups were weighed weekly and at the time of sacrifice. Body weight gains were calculated for males and females during each appropriate interval.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Although not a feeding study, food consumption was determined weekly during the premating period (no mating period) for all males and females. Individual food consumption was measured and recorded weekly thereafter for the females in the general toxicity groups and during the gestational period for the females in the reproductive groups. Food consumption was also recorded on LD0 and LD4. The data were then used to calculate food efficiency for the associated intervals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Data from food consumption were used to determine food efficiency for associated intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: See detailed clinical observations
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of treatment
- Anaesthetic used for blood collection: Yes. (Isoflourane anesthesia ) collected via orbital sinus bleeding.
- Animals fasted: Yes, 18 hours prior to blood collection
- How many animals: 5 males and 5 females/dose level
- Parameters examined: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, prothrombin time and activated partial thromboplastin time.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28 of treatment
- Animals fasted: Yes. 18 hours prior to blood collection
- How many animals: 5 males and 5 females/dose level
- Parameters examined: calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alanine aminotransferase (SGPT), serum aspartate aminotransferase (SGOT), gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin, total serum protein, globulin, total cholesterol, alkaline phosphatase and magnesium measurements.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters examined:

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the final days of treatment
- Dose groups that were examined: Five male and five females/dose group (including controls).
- Battery of functions tested: sensory activity / grip strength / motor activity: excitability, autonomic function, gait and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behavior) and other abnormal clinical signs including but not limited to convulsions, tremors, unusual or bizarre behavior, emaciation, dehydration, and general appearance. The rats were observed in random without the observer aware of the dose group.

Motor activity was also evaluated. Each animal was evaluated for a single one-hour phase, with photobeam counts accumulated over six, 10-minute intervals. Total movements (consisting of fine and active movements) was considered an appropriate measure for the assessment of potential behavior effects in this screening level study.)
Maternal examinations:
Necropsy: Gross necropsy of all males and females included an initial examination of
external surfaces and orifices, as well as the cranial, thoracic and abdominal cavities and
their contents. Rats were examined for gross lesions. Gross lesions were retained in
neutral buffered. 10% formalin (NBF).
• Reproductive Groups: Special attention was paid to the organs of the
reproductive system. For male rats, the testes and epididymides. seminal vesicles
with coagulating gland and prostate were retained. The testes and epididymides
were weighed. The testes were fixed in Bouin's solution for 4-12 hours
(depending on size) before being retained in 70% alcohol For females, special
care was taken to examine the uterus for the presence of conceptuses .. For each
female, the ovaries were weighed; the ovaries, uterus and accompanying
structures, and a mammary gland were retained in NBF. Subsequently, the fixed
uteruses from all adult female rats (including those that did not deliver litters and
those that delivered pups) were stained with potassium ferricyanide to confirm
pregnancy status and to determine post-implantation toss.

Rats were evaluated for gross lesions. Pregnancy status and uterine contents of
female rats were recorded.. Aborted fetuses and/or delivered pups were
examined to the extent possible. Organs and tissues were excised, weighed,
preserved and/or stained and implantation sites counted as described for 1hose
animals sacrificed by design.

• General Toxicology Groups: At scheduled sacrifice, all survivors were
euthanized by exsanguination from the abdominal aorta under isoflourane
anesthesia. All animals were subjected to a full necropsy that included
examination of the external surface of the body, all orifices and the thoracic.
abdominal and cranial cavities and their contents. The liver, kidneys, adrenals,
brain, heart, thymus, spleen. ovaries, testes and epididymides (of all animals
sacrificed by design) were weighed wet as soon as possible after dissection to
avoid drying. The following organs and tissues from all animals were preserved
in NBF for possible future histopathological examination: all gross lesions,
lungs, brain~ including sections of the medulla/pons. cerebellar cortex and
cerebral cortex, spinal cord (3 levels: cervical, mid-thoracic, and lumbar), eyes,
pituitary. thyroid/parathyroid, thymus, trachea, heart. sternum with bone marrow,
salivary glands, liver, spleen, kidneys, adrenals, pancreas, ovaries, testes, uterus
(with attached urinary bladder, cervix and vagina), accessory sex organs
(epididymides, prostate, and seminal vesicles). female mammary gland., skin.
aorta. esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum,
representative lymph node, and peripheral nerve (sciatic).

HISTOPATHOLOGY: Yes
Organs: Histopathologic examination was performed on the preserved organs and tissues of the Reproductive and General Toxicity Group animals from the control (Groups I and II) and high dose (Groups VII and VIII). In addition, gross lesions of potential toxicological significance noted in any test groups were also examined. Microscopic findings were graded.
Statistics:
Mean and standard deviations were calculated for all quantitative data. Except for clinical pathology data were the contract laboratory, Huntingdon Life Sciences, elected to use statistics to aid in the data interpretation; no further statistical treatment of the study was conducted due to small group sizes.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
see IUCLID 7.8.1 and 7.5.1
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Remarks on result:
not measured/tested
Abnormalities:
not specified
Developmental effects observed:
not specified

Reproductive subgroup: There were no treatment-related deaths and no signs of overt clinical toxicity. There were no effects on body weight, food consumption, or food efficiency. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring.

WATER CONSUMPTION AND COMPOUND INTAKE

There were no effects of test-substance treatment on food consumption in males. There were no effects of food consumption on females during pre-mating; during Weeks 3, 4, and 5 for females in the General Toxicity Group; or during gestation and lactation. Food consumption was not measured during the mating period. Food efficiency was also unaffected by treatment.

 

ORGAN WEIGHTS:

Mean organ weights and organ-to-body weight ratios for both the Reproduction and General Toxicity test groups, in general, were considered comparable to their respective control groups. Any slight increases or decreases from the control were incidental, not dose-related and judged not to be of toxicological importance.

 

GROSS PATHOLOGY:

There were a number of gross observations correlated to microscopic findings. The dilatation of the uterus (horns) observed in several female rats from the General Toxicology Group (Group II", Animal #8133 and 8134, Group VIII - Animal #8205, 8206, 8207 and 8208) was considered to be a function of the· estrus stage (generally proestrus, but sometimes early estrus). These gross observations and others, along with their microscopic correlates, were all considered incidental background findings not attributable to administration of the test substance.

Conclusions:
Based on the results of a combined repeated dose toxicity study with a reproduction/ developmental toxicity screening performed according to OECD 422 guideline and GLP principles, the NOAEL of potassium nitrate was found to be >= 1,500 mg/kg/day for developmental toxicity.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. However, since the study was performed with a substance analogue and the data are read across, the Klimisch score is 2.
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 reliable study with sodium nitrate is present.A reliable 28-day oral OECD 422 study has been performed in rats (5 rats/sex/dose) via gavage, containing 50, 750 or 1500 mg/kg bw/day potassium nitrate. There were no treatment-related deaths and no signs of overt clinical toxicity. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring. Therefore, it was concluded that the NOAEL is >=1500 mg/kg bw/day (or higher, highest dose tested).

Based on an expert statement, no further studies need to be conducted. No developmental effects were observed up to and including the highest dose tested of 1500 mg/kg bw/d in an oral OECD 422 study in rats with potassium nitrate. In a 2-generation study in dogs doses with up to 1000 mg sodium nitrate/L in the drinking water no indication of significant adverse effects on development, fertilization or hypothyroidism was observed. Sodium is an essential element for humans and the acceptable daily intake for sodium is 2.4 g/day (Dutch Voedingscentrum). In relation to this value exposure due to use as described under REACH is considered minimal.

Moreover, sodium nitrate has been used in fertilizers for over decades without an identifiable relation to reproductive or developmental effects.

JECFA and the former SCF derived an ADI of 3.7 mg/kg bw/d for nitrate, which was left unchanged by EFSA (EFSA, Nitrate in vegetables. Scientific opinion of the panel on contaminants in the food chain, 2008). This ADI was based on a 2-y study in rats with a NOEL of 500 mg sodium nitrate/kg bw/ (= 370 mg/kg bw/d nitrate)and a subchronic study with sodium nitrate in dogs with a NOEL of 370 mg/kg bw/d for nitrate. The OECD study with potassium nitrate in this dossier has a NOAEL of 1500 mg/kg bw/d which is equivalent to 62/101.1 x 1500 = 920 mg nitrate/kg bw/d, which is higher than the study on which the ADI was based. As the exposure assessment using this value indicates safe use, it is also considered safe for reproductive and developmental toxicity.

EFSA recently (June 2017) re-evaluated sodium nitrate and potassium nitrate as food additives. The Panel concluded that currently there was insufficient evidence to withdraw the ADI of 3.7 mg/kg bw/d for nitrate.

Justification for selection of Effect on developmental toxicity: via oral route:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles. However, since the study was performed with a substance analogue and the data are read across, the Klimisch score is 2. The read-across rationale is attached in the appropriate target study record.

Justification for classification or non-classification

According to Annex I of Regulation (EC) No. 1272/2008 sodium nitrate is not classified based on the available data:

- classification for reproductive toxicity is not required based on a NOAEL of 1500 mg of the read-across substance potassium nitrate per kg bw in a screening study and supporting evidence of studies with the substance showing the absence of toxicity;

- classification for developmental toxicity is not required based on a NOAEL of 1500 mg of the read-across substance potassium nitrate per kg bw.

An expert statement justifies the absence of data for reproductive toxicity and developmental toxicity and therefore the classification of the substance (not classified).

Additional information