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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2002-01-08 to 2002-02-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented GLP study. Study was performed according to guideine OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) with minor deviations (only 5 animals per sex tested instead of 10). The read-across rationale can be found in the category approach document attached in Section 13.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
5 animals of each sex instead of 10 animlas of each sex tested
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium nitrate
EC Number:
231-818-8
EC Name:
Potassium nitrate
Cas Number:
7757-79-1
Molecular formula:
HNO3.K
IUPAC Name:
potassium nitrate
Details on test material:
- Name of test material (as cited in study report): Potassium nitrate
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: solid
- Analytical purity: > 97%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: PSL Referenc number: 011101-3D (admin test days 1-31) and 020122-1D (admin test days 32-47)
- Expiration date of the lot/batch: None given: Supplied 2001-11-01 and again 2002-01-22
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling) not applicable:
- Stability under test conditions:Stable
- Storage condition of test material: Room temperature
- Other: no data

Test animals

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: no data
- 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
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): Purina Certified Rodent Diet #5002; ad 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 °C
- Humidity (%): 43-66%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 2002-01-08 To: 2002-02-23

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
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 targeted dose level for all rats in the General Toxicity groups (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 stirr plate while being sampled to dose the test animals during the study.

DIET PREPARATION:
- Rate of preparation of diet (frequency): not applicable, route of administration of test substance is via gavage
- Mixing appropriate amounts with (Type of food): not applicable
- storage temperature of food: no data
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 µg/mL), supplied by EM Science, was used for calibration.
Duration of treatment / exposure:
daily
Dose frequency: each animal was dosed by oral intubation using a stainless steel ball-tipped gavage needle attached to an appropriate syringe. Dose administration was daily (7 days/week) for all adult animals as follows:
Male: general toxicity groups: for at least 28 days of exposure
Female rats: for at least 28 days of exposure
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 750 and 1500 mg/kg/day
Basis:
other: Doses were selected based on parameters assessed in a range-finding study at concentrations up to 1000 mg/kg/day)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
other: yes, but no data mentioned

Examinations

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 gestional days (GD7, GD14 and GD20) and lactational (LD4 only) periods. Female rats were evaluated for adverse clinical signs during parturition. Maternal behaviour was checked on LD0 and LD4 and recorded. The date and clock time of all observations and/or mortality 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:
Data from food consumption were used to determine food efficiency for associated intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: See detailed clinical observations
- Dose groups that were examined: No data

HAEMATOLOGY: Yes (collected via orbital sinus bleeding)
- Time schedule for collection of blood: Day 28 of treatment
- Anaesthetic used for blood collection: Yes: Isoflourane anesthesia
- Animals fasted: Yes: 18 hours prior to blood collection
- How many animals: 5 males and 5 females/dose level
- Parameters checked: 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 checked: 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
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the final days of treatment
- Dose groups that were examined: Five male and 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 behaviour) and other abnormal clinical signs including but not limited to convulsions, tremors, unusual or bizarre behaviour, 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) were considered an appropriate measure for the assessment of potential behaviour effects in this screening level study).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: 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). 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 weiged 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: histopathological 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.

Results and discussion

Results of examinations

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 specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
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: there 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.

FOOD 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: Food efficiency was also unaffected by treatment.

WATER CONSUMPTION AND COMPOUND INTAKE: not applicable

OPHTHALMOSCOPIC EXAMINATION: no effects

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 1500 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 or renal dysfunction (e.g., creatinine) discounted the clinical siginificance 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 1500 mg/kg/day. These changes were within the range of historical control and were not considered to be of biological significance.

URINALYSIS: no data

NEUROBEHAVIOUR: Functional observational battery (FOB) and motor activity tests identified no treatment-related changes in behaviour, 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 (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 identical background findings not attributable to administration of the test substance.

HISTOPATHOLOGY: No treatment-related histopathological changes were reported.

HISTORICAL CONTROL DATA (if applicable): no data

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were seen on general toxicity endpoints.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

NOAEL: 1500 mg/kg/day (general toxicity)

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

Applicant's summary and conclusion

Conclusions:
A NOAEL of 1500 mg/kg/day for general toxicity was derived after exposure to potassium nitrate. NO adverse effects were seen on general toxicity endpoints.