1-nitroguanidine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: LAIR: OP-STX-74

Deviations:

yes

Remarks:

The dosing phase was accomplished according to the protocol with the following exemption: Food consumption data collected on 13 September 1985 (Week 3) for animals 85D00858 through 85D00906 were lost due to a computer malfunction. Food consumption and bod

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA 560/6-82-001

Deviations:

yes

Remarks:

The dosing phase was accomplished according to the protocol with the following exemption: Food consumption data collected on 13 September 1985 (Week 3) for animals 85D00858 through 85D00906 were lost due to a computer malfunction. Food consumption and bod

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP Compliance for LAIR study 85042

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-nitroguanidine

EC Number:

209-143-5

EC Name:

1-nitroguanidine

Cas Number:

556-88-7

Molecular formula:

CH4N4O2

IUPAC Name:

N-nitroguanidine

impurity 1

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

Na2O4S

IUPAC Name:

disodium sulfate

impurity 2

Reference substance name:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

impurity 3

Reference substance name:

4,6-diamino-1,3,5-triazin-2(1H)-one

EC Number:

211-455-1

EC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

Cas Number:

645-92-1

Molecular formula:

C3H5N5O

IUPAC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

impurity 4

Reference substance name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

EC Number:

211-456-7

EC Name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

Cas Number:

645-93-2

Molecular formula:

C3H4N4O2

IUPAC Name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin Kingman, Fremont, CA
- Age at study initiation: young adults
- Weight at study initiation: 95 - 264 g
- Acclimatisation period: 14 or 16 days (males and females respectively)
- Housing: clear, polycarbonate show boxes in drawner rack cageswith Alpha-dri bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 27.0 °C
- Humidity (%): 31-50%
- Photoperiod (hrs dark/hrs light): 12/12 with a 0.5 hours dawn phase in and a 0.5 hours dusk phase out.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Mixing appropriate amounts with (Type of food): Purina Rodent Chow 5002 Meal Form (Ralston Purina, St. Louis, MO)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chromatographic Analysis (HPLC)
All diet preparations were done in accordance with LAIR SOP OP-STX-16. All diet mixes were within 6.4% of target concentrations and were adequately homogenous.

Duration of treatment / exposure:

90 days

Frequency of treatment:

ad libitum

Doses / concentrations

Remarks:

Doses / Concentrations:
1.05 to 21.19 mg NiGu/g diet
Basis:
nominal in diet

No. of animals per sex per dose:

15

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: basis were the results of an acute toxicity study and a 14-day subacute study. The acute oral median lethal dose exceeded a LIMIT dose of 5000 mg/kg. Thus, the upper dose level used in the subacute study was a limit dose of 1000 mg/kg. Doses were selceted on basis of a logarithmic progeresssion table.
- Rationale for animal assignment (if not random): random (weight bias, stratified animal allocation)

Positive control:

no positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study); WATER CONSUMPTION:
- Measured on a twice weekly basis
- Individual feed jars were used
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes



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: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.1] were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes:
No compoud-related gross or microscopic lesions were observed. All gross and microscopic lesions were minimal to mild in severity and considered to be incidental findings commonly observed in Sprague-Dawley rats. There were no microscopic lesions that were significantly different in severity from the control using the Kolgorov-Smirnov two-tailed test.

HISTOPATHOLOGY: Yes

Statistics:

Animal weights, food consumption, and water consumption
Equality of variances, (Levene's Test), standard one-way analysis of variance (ANOVA) / Welch one-way ANOVA, F-statistic, Kruskal-Wallis one-way ANOVA for bilirubin values,
organ weights, haematology, and serum chemistry using standard one-way ANOVA
Bartlett's test for homogeneity of groups, modified t-test / Dunnett's test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occurred during the study.
No clinical signs of toxicity attributable to nitroguanidine administration were observed during the 90-day study period.

BODY WEIGHT AND WEIGHT GAIN
No significant differences from controls were exhibited in the male dose groups. The females in the 1000 mg/kg/day group exhibited a statistically significant (p<=0.05) decrease in the rate of growth when compared to the controls on Weeks 5, 6, 8, 9, and 12. During the course of the study, the female control group mean weight gain was 103 g while the female high-dose group mean weight gain was only 81 g.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in the males increased in all dose groups during the 13 weeks of the study. The rate of the increase in food consumption was not dose related. Food consumption in the 1000 mg/kg/day groups was significantly (p<= 0.05) lower than in the control group during Week 1 for the males, and weeks 5 and 6 for the females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption by both sexes increased in a dose-related manner during the study. For the 316 and 1000 mg/kg/day groups this increased water consumption was significant.

HAEMATOLOGY
No consistent treatment-related changes were noted in serum chemistry or haematological values.

CLINICAL CHEMISTRY
No significant (p<=0.05) differences from controls were observed in electrolyte levels for either sex at any of the dose groups. The group 4 males exhibited mean cholesterol values significantly (p<=0.05) less than those of the controls of the controls at interim and terminal sacrifice, but these values remained within normal limits. The group 3 males exhibited mean LDH and total protein values significantly (p<=0.05) less than those of the controls at terminal sacrifice, but the values remained within normal limits. The group 2 and group 4 females exhibited mean triglyceride values significantly (p<=0.05) less than those of the controls at terminal sacrifice, but these values remained within normal limits.

ORGAN WEIGHTS AND RATIOS
Organ weight, organ-to-body weight ratios, and organ-to-brain weight ratios were compared for liver, spleen, adrenals, kidneys, heart, testes/ovaries, and brain. The group 3 males showed a significant (p<=0.05) decrease in absolute adrenal gland weight as compared to controls at interim sacrifice. At terminal sacrifice the males exhibited a loss in heart weight that appeared to be dose related but not statistically significant (p<=0.05). The females showed significant (p<=0.05) decreases in absolute ovarian weights in the 100, 316, and 1000 mg/kg groups at interim sacrifice, decreased brain weight at interim sacrifice in the 1000 mg/kg group and a decreased spleen weight in the 316 mg/kg group at term sacrifice. The spleen-to-brain weight ratios for the 100 and 316 mg/kg female groups were significantly (p<=0.05) decreased compared to that of the controls at terminal sacrifice.

GROSS PATHOLOGY AND HISTOPATHOLOGY: NON-NEOPLASTIC
No compound-related gross or microscopic lesions were observed. All gross and microscopic lesions were minimal to mild in severity and considered to be incidental findings commonly observed in Sprague-Dawley rats. There were no microscopic lesions that were significantly different in severity from the control using the Kolmogorov-Smirnov two-tailed test.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Nitroguanidine, fed at dose levels from 100 mg/kg/day to 1000 mg/kg/day in the diet for 90 days, did not cause any appreciable toxicological effects under the conditions of this study.

Executive summary:

The 90-day subchronic oral toxicity of nitroguanidine was evaluated in male and female Sprague-Dawley rats. Nitroguanidine was administered in the diet at dose levels of 0, 100, 316, and 1000 mg/kg/day for 90 days. The addition of nitroguanidine to the diet consistently reduced food consumption and caused significant (p<= 0.05) increase in water consumption. Significantly (p <= 0.05) reduced weight gains were observed in the female high-dose group for 5 of the 13 weeks of the study period. No other clinical signs attributable to the test compound were observed during the study. Blood samples taken at necropsy for haematological and serum chemistry analyses exhibited no significant (p <= 0.05) abnormalities that could be attributed to nitroguanidine dosing. Microscopic examination of tissues from the control and 1000 mg/kg/day dose group animals revealed no lesions attributable to the administration of nitroguanidine. These findings indicate that nitroguanidine is nontoxic in rats when administered at doses as high as 1000 mg/kg/day for 90 days. The findings of increased water consumption suggest that nitroguanidine, which is excreted unchanged in the rat's urine, may be acting as an osmotic diuretic.