Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 7, 2002 – October 14, 2002
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
The study was performed with a substance analogue and the data are read across.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: OECD 422
not specified
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium nitrate
EC Number:
EC Name:
Potassium nitrate
Cas Number:
Molecular formula:
potassium nitrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Potassium Nitrate

Test animals

Details on test animals or test system and environmental conditions:
- 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

- 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

Administration / exposure

Route of administration:
oral: gavage
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:
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:
Duration of test:
tox group: 28 days
repro group: 53 days
Doses / concentrations
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)
other: Doses were selected based on parameters assessed in a range-finding study at concentrations up to 1,000 mg/kg/day
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- 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.

- 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.

- 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.

- 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.

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

- 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.
- 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:

- 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).

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.
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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
see IUCLID 7.8.1 and 7.5.1

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
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:
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.


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.



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.



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.

Applicant's summary and conclusion

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.