Ammonium nitrate

Endpoint summary

Description of key information

No repeated dose study is available for ammonium nitrate via the oral route. However, based on an oral OECD 422 study with potassium nitrate a NOAEL of ≥1500 mg/kg bw/day was derived. In addition, subchronic and chronic studies with ammonium sulphate have been included, to investigate the effect of the cation ammonium on the repeated dose toxicity. Based on these studies a NOAEL of 256 mg/kg bw/day was derived for chronic toxicity. The read-across rationale can be found in the category approach document attached in the target study record.

Because the particle size distribution shows that no inhalable fraction of the substance is present (0%<200 micrometer) and the vapour pressure is negligible, inhalation is not a likely route of exposure. Therefore, no repeated dose toxicity study is required. Two studies are available showing no toxic effects. A NOAEC of >= 185 mg/m^3 air was determined.

An expert statement is provided based on all scientific information available, which concludes that no additional sub-chronic toxicity studies need to be performed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented literature. The study has been performed in line with OECD and/or EC guidelines but with a substance analogue and the data are read across.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

In this study, the chronic toxicity (52-week oral feeding) and carcinogenicity (104-week oral feeding) was investigated.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Yoneyama Kagaku Kogyo (Osaka Japan)
- Age at study initiation: 5 weeks
- Housing: three or four rats per plastic cage
- Diet: ad libitum
- Water: Tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 °C
- Humidity (%): 55 ± 5 %
- Air changes (per hr): 18
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Recapture rates for ammonium sulfate from the admixed diet at each concentration level were confirmed to be 95.4-98.7%.

Duration of treatment / exposure:

52 and 104 weeks

Frequency of treatment:

continuously in the diet

Remarks:

Doses / Concentrations:
42, 256, 1527 mg/kg bw/day (males); 48, 284, 1490 mg/kg bw/d (females
Basis:
other: actual ingested, 52-week chronic toxicity study

Remarks:

Doses / Concentrations:
0.1, 0.6, 3.0% in the diet
Basis:
other: nominal in diet, 52-week chronic toxicity study

Remarks:

Doses / Concentrations:
564.1, 1288.2 mg/kg bw/d (males); 649.9, 1371.4 mg/kg bw/d (females)
Basis:
other: actual ingested, 2-year carcinogenicity study

Remarks:

Doses / Concentrations:
1.5, 3 % in the diet
Basis:
other: nominal in diet 2-year carcinogenicity study

No. of animals per sex per dose:

Chronic toxicity study: 10/sex
Carcinogenicity study: 50/sex

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks until week 10 and every 5 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 52 weeks
- Anaesthetic used for blood collection: Yes: ether
- Animals fasted: Yes: overnight
- How many animals: 10
- Parameters checked: red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Plt) and white blood cell count (WBC). Differential leukocyte counts and the reticulocyte count (Ebl).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 52 weeks
- Animals fasted: Yes: overnight
- How many animals: 10
- Parameters checked: total protein (TP), albumin (Alb), albumin/globulin ratio (AIG), total bilirubin (T-bil), total cholesterol (T-Cho), triglyceride (TG), blood urea nitrogen (BUN), creatinine (Cre), calcium (Ca), inorganic phosphorus (IP), sodium (Na), potassium (K), chloride Cl), aspartate transaminase (AsT), alanine transaminase (AlT), alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Brain, lungs, heart, spleen, liver, adrenals, kidneys and testes were weighed. As for adrenals, kidneys and testes, weights of each side were separately recorded and the total of both sides was used for calculation of group mean and SD values.
In addition to these organs, the nasal cavity, trachea, aorta, pituitary, thyroids, parathyroids, salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, pancreas, urinary bladder, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, mammary glands, skin, mesenteric and submandibular lymph nodes, thymus, sternum, femur including bone marrow, sciatic nerve, trigeminal nerve, spinal cord (cervical, thoracic and lumber cords), eye, Harderian gland and thigh muscle. All organs and tissues in the control and 3.0% group animals were histopathologically examined. Additionally, macroscopically abnormal sites in the 0.1% and 0.6% group animals in the chronic study and all organs and tissues of the 1.5% animals in the carcinogenicity study were also histopathologically examined.

Statistics:

Variance in data for body weights, hematology, serum biochemistry and organ weights were checked for homogeneity by Bartlett test. When the data were homogeneous, one-way analysis of variance (ANOVA) was used. In the heterogeneous cases, the Kruskal-Wallis test was applied. When statistically significant differences were indicated, Dunnett's multiple test was employed for comparison between control and treated groups. Final survival rates and the incidences of tumor and non-neoplastic lesions were compared with the Fisher's exact probability test or the Mann-Whitney's U-test.

Details on results:

CLINICAL SIGNS AND MORTALITY
In the chronic toxicity study, no mortality was found in any groups throughout the treatment period.
In the carcinogenicity study, the survival rate of control, 1.5% and 3.0% groups were 88%, 78% and 76%, respectively, for males, and 76%, 80% and 80%, respectively, for females, and no significant differences were observed among the groups.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related change in the body weights was found.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A tendency for increase of food intake in the male 3.0% group in the chronic toxicity study was noted.

HAEMATOLOGY
No significant variation was found.

CLINICAL CHEMISTRY
No dose related alteration was found.

ORGAN WEIGHTS
Absolute and relative kidney weights were increased or showed a tendency for increase at 3.0% in both sexes in the chronic toxicity study. Absolute spleen weights were decreased and relative liver weights were increased in the 3.0% male dose group. No dose-related changes were found in the other organs.

GROSS PATHOLOGY
There were no obvious macroscopic findings in any group in either the chronic toxicity or carcinogenicity studies, except for massive, nodular or focal lesions suggesting neoplastic change in the carcinogenicity study.

HISTOPATHOLOGY:
In the carcinogenicity study, non-neoplastic and neoplastic lesions were noted in the control and treatment groups, with no significant inter-group difference in their incidences or severity.

Key result

Dose descriptor:

NOAEL

Effect level:

256 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: 0.6% in the diet; Absolute and relative kidney weights were increased at the high dose level for both sexes. Absolute spleen weights were decreased and relative liver weights were increased in high dose males.

Dose descriptor:

NOAEL

Effect level:

284 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: 0.6% in the diet: Absolute and relative kidney weights were increased at the high dose level for both sexes.

Critical effects observed:

not specified

Organ weight of male rats fed diet containing ammonium sulfate for 52 weeks (Chronic toxicity study).

	control	0.1%	0.6%	3.0%
Body weight (g)	410.9 ± 12.3	428.6 ± 17.6	416.7 ± 23.7	400.5 ± 15.1
Brain (g)	2.04 ± 0.05	2.03 ± 0.07	2.05 ± 0.05	2.04 ± 0.05
Lungs (g)	1.20 ± 0.09	1.23 ± 0.21	1.16 ± 0.07	1.13 ± 0.06
Heart (g)	1.09 ± 0.08	1.10 ± 0.07	1.08 ± 0.05	1.08 ± 0.07
Spleen (g)	0.73 ± 0.05	0.72 ± 0.04	0.83 ± 0.36	0.68 ± 0.04 *
Liver (g)	9.62 ± 0.58	9.92 ± 0.73	10.26 ± 0.63	10.0 ± 0.85
Adrenals (g)	0.03 ± 0.01	0.04 ± 0.01	0.04 ± 0.00	0.04 ± 0.00
Kidneys (g)	2.35 ± 0.25	2.32 ± 0.11	2.42 ± 0.11	2.51 ± 0.11 *
Testes (g)	3.38 ± 0.17	3.27 ± 0.11	3.25 ± 0.25	3.29 ± 0.14

Organ weight of female rats fed diet containing ammonium sulfate for 52 weeks (Chronic toxicity study).

	control	0.1%	0.6%	3.0%
Body weight (g)	207.4 ± 13.49	220.3 ± 8.68	219.2 ± 13.62	212.7 ± 24.39
Brain (g)	1.86 ± 0.04	1.83 ± 0.04	1.83 ± 0.05	1.82 ± 0.05
Lungs (g)	0.82 ± 0.06	0.79 ± 0.10	0.83 ± 0.12	0.79 ± 0.05
Heart (g)	0.65 ± 0.05	0.67 ± 0.05	0.70 ± 0.03	0.67 ± 0.05
Spleen (g)	0.44 ± 0.04	0.44 ± 0.02	0.45 ± 0.03	0.45 ± 0.07
Liver (g)	4.44 ± 0.26	4.66 ± 0.35	4.69 ± 0.40	4.89 ± 0.42
Adrenals (g)	0.04 ± 0.00	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.01
Kidneys (g)	1.25 ± 0.07	1.35 ± 0.08 *	1.35 ± 0.09	1.39 ± 0.08 **

* Significantly different from the control at p<0.05. **Significantly different from the control at p<0.01.

Executive summary:

In the subchronic part of the study, groups of 10 rats/sex were fed a diet containing the test substance (purity not given) at concentrations of 0, 0.1, 0.6, or 3% for 1 year. These concentrations corresponded to average daily intakes of 0, 42, 256, and 1527 mg/kg bw/d for males and 0, 48, 284, and 1490 mg/kg bw/d for females, respectively. For investigation of the carcinogenic potential, groups of 50 rats/sex were fed a diet containing the test substance (purity not given) at concentrations of 0, 1.5, or 3% for 2 years. These concentrations corresponded to average daily intakes of 0, 564.1, and 1288.2 mg/kg bw/d for males and 0, 649.9, and 1371.4 mg/kg bw/d for females respectively.

Absolute and relative kidney weights were increased at the high dose level for both sexes. Absolute spleen weights were decreased and relative liver weights were increased in high dose males. No macroscopic changes were recorded by gross pathology, except for massive nodular or focal lesions suggesting neoplastic changes. At histopathological examination, non-neoplastic and neoplastic lesions were noted in the control and treatment groups, with no significant inter-group difference in their incidences or severity.

The authors concluded that the no observed adverse effect level of ammonium sulfate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg bw/d in males and females, respectively, and the compound is noncarcinogenic under the conditions of the study.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

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)

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 oral 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.

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

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

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

Observations and examinations performed and frequency:

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: Yes: 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: 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.

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.)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes
HISTOPATHOLOGY: Yes
- 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).

-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

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

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: General toxicity: no adverse effects (highest dose tested)

Critical effects observed:

not specified

Toxicity 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. Functional observational battery (FOB) and motor activity tests identified no treatment-related changes in behavior, function, or motor activity. 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. No treatment-related histopathological changes were reported.  

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 general 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. Based on these data, the NOAEL of potassium nitrate was found to be >=1,500 mg/kg/day for general toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

256 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. The read-across rationale can be found in the category approach document attached in Section 13.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The report was very limited documented, although the analytical part was more extensive. Also limited parameters were observed.

Principles of method if other than guideline:

No data

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

inhalation

Type of inhalation exposure:

nose/head only

Vehicle:

other: no data

Remarks on MMAD:

MMAD / GSD: particles < 5 microm. = between 26 and 50 wt%.
30% of the particles were < 10 micrometer

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

5 hrs/day; 5 days/week

Dose / conc.:

185 mg/m³ air (analytical)

Remarks:

Maximum dose
Minimum dose: 26 mg/m^3 air

No. of animals per sex per dose:

10 animals were exposed to ammonium nitrate

Control animals:

other: yes, 5 control animals were exposed to an air current, 5 control animals were totally untreated.

Observations and examinations performed and frequency:

Bodyweights were registered before, during and after exposure.

Sacrifice and pathology:

After killing, autopsy was carried out and organs were taken for histopathological analysis.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

>= 185 mg/m³ air

Sex:

male

Basis for effect level:

other: No effects observed.

Critical effects observed:

not specified

A certain irritation in the nostrils of the test group was seen, no further symptoms. However, it was not clear at which concentrations these effects were seen. No significant differences in body weight between test and control animals. No macroscopically or microscopically effects have been seen. Post exposure observation period: 3 days

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

185 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The report was very limited documented, although the analytical part was more extensive. Also limited parameters were observed.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

No oral repeated dose toxicity studies with ammonium nitrate are available.

A 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. There were no effects on body weight, food consumption, or food efficiency. No effects on neurobehaviour were observed. No treatment-related histopathological changes were reported. Therefore, it was concluded that the NOAEL is 1500 mg/kg bw/day (or higher, highest dose tested).

Further repeated dose toxicity studies were present with ammonium sulfate. As it is shown from the data available for all nitrate substances in the inorganic nitrate category, repeated dose toxicity is very low with regard to systemic effects. As ammonium nitrate has a cation, ammonium, that might play a role in repeated dose toxicity, also studies of substances with ammonium as cation are described here.

In a 13-week study rats (10/sex/dose) were exposed to diet containing 0, 0.38, 0.75, 1.5 or 3 % ammonium sulfate (corresponding to 0, 222, 441, 886, 1792 mg/kg bw/day in males and to 0, 239, 484, 961, 1975 mg/kg bw/day in females). No substance-related changes were found in body weights, haematology and serum parameters, or in the histological examinations (brain, heart, lung, liver, kidney, adrenal gland, spleen, testes, thymus). The relative testes weight was significantly increased at all doses, but no histological effects were found, not considered to be treatment related. Male animals of the highest dose group exhibited diarrhea during the administration period. According to the authors the NOAEL (male) was 886 mg/kg bw/day and the NOAEL (female)was 1975 mg/kg bw/day. 

A chronic oral toxicity and carcinogenicity study was conducted in rats, similar to the requirements of OECD TG 453. In the subchronic part of the study, groups of 10 rats/sex were fed a diet containing the ammonium sulfate (purity not given) at concentrations of 0, 0.1, 0.6, or 3% for 1 year. These concentrations corresponded to average daily intakes of 0, 42, 256, and 1527 mg/kg bw/d for males and 0, 48, 284, and 1490 mg/kg bw/d for females, respectively. For investigation of the carcinogenic potential, groups of 50 rats/sex were fed a diet containing the test substance (purity not given) at concentrations of 0, 1.5, or 3% for 2 years. These concentrations corresponded to average daily intakes of 0, 465.1, and 1288.2 mg/kg bw/d for males and 0, 649.9, and 1371.4 mg/kg bw/d for females respectively.Absolute and relative kidney weights were increased at the high dose level for both sexes. Absolute spleen weights were decreased and relative liver weights were increased in high dose males. No macroscopic changes were recorded by gross pathology, except for massive nodular or focal lesions suggesting neoplastic changes. At histopathological examination, non-neoplastic and neoplastic lesions were noted in the control and treatment groups, with no significant inter-group difference in their incidences or severity.The authors concluded that the no observed adverse effect level of ammonium sulfate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg bw/d in males and females, respectively, and the compound is noncarcinogenic under the conditions of the study. There was no evidence of a long-term carcinogenic activity of the test substance.

Based on an expert statement, no additional studies with Ammonium nitrate are performed. In the 28-day study with potassium nitrate no toxicity was observed at the limit dose value of 1500 mg/kg bw/day.

The NOAEL of 1500 mg/kg bw/d 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. Based on the assessment factors for exposure duration (see guidance IR CSA R.8) from subacute to semi-chronic a factor 3 is used, expecting a 3 times lower NOAEL. Assuming this factor would be applicable for this substance, this would still not result in a higher NOAEL than the NOEL on which the ADI is based. In addition, EFSA recently (June 2017) re-evaluated sodium nitrate and potassium nitrate as food additives taking all chronic toxicity and carcinogenicity studies available into account. It was concluded that there is currently insufficient evidence to withdraw the ADI of 3.7 mg/kg bw/d for nitrate.

Dermal

No dermal studies are present.

Inhalation

Two limited inhalation studies with ammonium nitrate were present. In one study rats and guinea pigs were exposed for 4 weeks to 1 mg/m3, 5 days/week, 6 hrs/day, showing no effects on body weight, lung volume, vital capacity, histologic structure of the respiratory tract. In another study rats were exposed up to 185 mg/m3 for 2 weeks for 5 hrs/day, 5 days/week, which did also not show any effect. Only bodyweights were recorded and histopathological examinations were performed. However, testing concentrations were very low with no effect at the higest dose tested: NOAEC ≥1 mg/m3 and NOAEC ≥185 mg/m3 (unclear whether this is the maximum attainable concentration). Therefore, the oral data are considered to be of more value with regard to risk assessment.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

One 28-day study with the read-across substance potassium nitrate is available, in addition one sub-chronic and one chronic key study with ammonium sulfate is present. The latter is used for DNEL derivation.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Two supporting inhalation studies are available on ammonium nitrate which showed no effects at the concentrations tested.

Justification for classification or non-classification

Based on the available data, ammonium nitrate does not have to be classified according to Regulation (EC) No 1272/2008 for repeated dose toxicity.