1-nitroguanidine

Endpoint summary

Description of key information

A subacute oral toxicity study (14 days) in rats and two subchronic oral toxicity studies (90 days) in mice and rats,

all according U.S. EPA 560/6-82-001, are available. No compound related adverse effects have been identified.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

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.

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

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

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

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.

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

Key result

Critical effects observed:

not specified

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.

 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - July 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: LAIR OP-STX-74

Qualifier:

according to guideline

Guideline:

other: U.S. EPA 560/6-82-001

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Indianapolis, IN
- Age at study initiation: 33 days
- Weight at study initiation: 17-23 g
- Housing: individually in clear, polycarbonate shoe boxes in drawer rack cages with alpha-Dri, a cellulose fibre, used as bedding
- Diet (e.g. ad libitum): ad libitum, certified Purina Rodent Chow 5002 Meal Form
- Water (e.g. ad libitum): 16-ounce water bottles with stoppers and sipper tubes
- Acclimation period: 14-15 days males and females respectively


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-25°C
- Humidity (%): 26-60%, occasionally up to 80% for several hours
- Photoperiod (hrs dark/hrs light): 12/12 with 0.5 hours dawn phase in and 0.5 hours dusk phase out

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Initially, stock solutions of nNGu (1mg/ml) and MNGu (1 ml/mg) in water were prepared. Standard solutions for the calibration plot were then prepared as dilutions to 25 ml with water of the stock solutions. These standards were analyzed at the beginning and end of each run. The stability of these solutions was verified by a stability study conducted during a 14-day oral subchronic toxicity study. Samples from the feed mixtures and premix were extracted by adding varying amounts of water and the MNGu stock solution (1 ml/mg). The samples were stirred for one hour. The supernatant from each tube was filtered through a Swinney adaptor with a millipore filter (0.2µm). This filtrate was subsequently analyzed using a Hewlett-Packard 1090 HPLC system.
To determine the homogeneity of the feed mixtures, samples were taken from the right, left, and botttom ports of the Twin Shell Blender used in preparation of the diet and analyzed. Samples for testing homogeneity were collected during the first and eighth weeks of the study.
The ratio of NGu and MNGu peak area was calculated for each of the standards and samples. Least squares linear regression analysis of the standard concentrations vs. the peak area rations was performed to obtain a standard curve. The curve used was in the form of the best fitting line. All calculations were performed on a TI-55 scientific calculator. To calculate the concentration in the diet in terms of mg of NGu per gram of diet, the concentration of the extract was multiplied by the dilution factor and divided by the weight of the diet sample extracted. The experimental concentrations were compared to the target concentrations and reported as a percent of target.

DIET PREPARATION
- Rate of preparation of diet (frequency): each week
- Mixing appropriate amounts with (Type of food): Certified Purina Rodent Chow 5002 Meal Form
- Food stored at temperature: 4°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The plots of NGu concentration versus peak area ratio were linear within the range of concentrations analyzed.
Under the conditions of the analysis, NGu eluted with a retention time of approximately 4.8 minutes, and MNGu eluted with a retention time of approximately 6.0 minutes. All samples (96) were analyzed within one week. Only one sample was extracted and analyzed from each batch of feed. The results from the analysis were recorded in the study.
The concentration of NGu in the diet mixtures was within 10% of the target concentration. Samples collected furing the first and eighth weeks of the study showed that the NGu concentration was homogeneous in the feed over the range tested, according to the EPA criteria for homogeneity.

Duration of treatment / exposure:

90 days

Frequency of treatment:

ad libitum

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

316 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in diet

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of an acute toxicity study and a pilot study.
- Rationale for animal assignment (if not random): Allocation was accomplished using a computer-based, stratified, weight biased randomization method (LAIR SOP OP-ISG-24).
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean weekly 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: No data

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All animals were subjected to a complete gross necropsy following exsanguination under sodium pentobarbital anesthesia. Full histopathology was performed on the organs and tissues listed in Appendix P (of the original study report) of all mice in the control and high-dose group. In addition, histopathology was performed on the brain, lungs, liver, kidneys, urinary bladder, and gall bladder of all study animals.

Statistics:

The animal weights, food consumption, and water consumption were analyzed statistically with packaged programs available on BMDP software. The equalit of the variances of the groups was tested using the Levene's Test . If the variances were equal, the vehicle control group and the dose groups were compared by the standard one-way analysis of variance (ANOVA). Otherwise, the Welch one-way ANOVA, which is not based on the assumption that the variances are equal, was performed. If the F-statistic was significant in either case, the Dunnett's test was performed to determine whether or not the vehicle control group was significantly different from any of the dose groups. Their was insufficient bilirubin data to perform statistical analysis. Statistical analysis for organ weights, hematology and serum chemistry were done on the Xybion software program using the standard one-way ANOVA. The homogeneity of the groups was tested by the Bartlettl's test. If the groups were found to be non-homogeneous, then a modified t-test was performed instead of the Dunnett's test.

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:

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

not examined

Details on results:

Water consumption:
Water consumption increased significantly (p ~0.05) in the male 1000 mg/kg/day group for weeks 1 through 13 when compared to the controls. The 100 mg/kg/day males showed a significant decrease in water consumption for week 6 when compared to the controls (p ~0.05).
Nitroguanidine may be acting as an osmotic diuretic in this study. Urea, a chemically related compound, has been used as an osmotic diuretic. Since nitroguanidine is considerably less soluble in water than guanidine or urea, the excretion of nitroguanidine in the urine would require considerably more urinary volume than would be required to excrete a similar quantity of guanidine or urea. The dose-related increase in water consumption following nitroguanidine administration observed in this study are consistent with an increased urinary volume requirement for excretion of nitroguanidine.

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

Key result

Critical effects observed:

not specified

Mortalities

No deaths occurred during the study.

 

Food, Nitroguanidine, and Water Consumption

No statistically significant (p ~0.05) dose or compound-related effects were observed in food consumption. Nitroguanidine consumption in the food ranged from 77 to 1211 mg/kg/day.

Water consumption increased significantly (p ~0.05) in the female 1000 mg/kg/day group for weeks 1 and 2. Water consumption increased significantly (p ~0.05) in the male 1000 mg/kg/day group for weeks 1 through 13 when compared to the controls. The 100 mg/kg/day males showed a significant decrease in water consumption for week 6 when compared to the controls (p ~0.05).

 

Body Weights

No statistically significant dose or compound related effects were observed in male or female body weights.

 

Clinical Signs

No clinical signs attributable to nitroguanidine administration were observed. The incidence of signs such as irritability, aggressive behavior, ocular changes, alopecia, hyperactivity, chewing, dehydration, twitching, and increased startle reflex appeared to be random, often isolated occurrences, and a function of a subchronic study rather than compound or dose related.

 

Clinical Chemistry

The group-4 males had a significantly increased mean aspartate amino-transferase (AST) level compared to that of the controls (p ~0.05) at interim sacrifice. The AST value was within normal limits compared to the baseline sacrifice performed at the beginning of the study period (Appendix Q). At terminal sacrifice, the group-3 males had significantly decreased uric acid levels compared to those of the controls (p ~0.05). However, the uric acid level was within normal limits. The interim sacrifice groups-3 and -4 females had significantly increased albumin and albumin-globulin ratio values compared to those of the controls (p ~0.05), but the values remained within the respective normal ranges established by the baseline sacrifice.

 

Hematology

No statistically significant variances from the controls were found in either the male or female dose groups.

 

Organ Weights and Ratios

Organ weights, organ-to-body weight ratios, and organ-to-brain weight ratios were compared for liver, spleen, kidneys, heart, testes/ovaries, and brain. The interim sacrifice group-4 males had a significantly greater brain to body weight ratio than the controls (p ~0.05). The group-4 male animals in general had greater absolute brain weights and smaller body weights than the other groups, but these differences were not statistically significant (p ~0.05). No statistically significant differences from the control were observed in the organ weights or organ weight ratios in the female dose groups.

 

Necropsy

No compound-related gross or microscopic lesions were observed. All gross and microscopic lesions were mild to minimal in severity and considered to be incidental findings commonly observed in aging mice. The 316 mg/kg/day female group had significantly decreased extramedullary hematopoiesis in the liver compared to the controls (p ~0.05), but this was considered an incidental finding not related to dosing. 

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 toxicologic effects, other than increased water consumption in the high-dose group, under the conditions of this study.

Executive summary:

In a subchronic toxicity study Nitroguanidin (99%) was administered to 15 ICR mice/sex/dose in diet at dose levels of 0, 100, 316, 1000 mg/kg bw/day.

No compound related effects in mortality, clinical signs, body weight, food consumption, hematology, or gross and histologic pathology were observed. Several serum chemistry parameters did exhibit statistically significant (p<= 0.05) alterations from the control values, but these changes were isolated occurrences with no consistent dose-dependency. With the exception of the brain-to-body weight ratio in the high-dose males, organ weights and their respective ratios were not significantly affected by dosing. The findings of increased water consumption suggest that nitroguanidine, which is excreted unchanged in the mouse's urine, may be acting as an osmotic diuretic.

 

The NOAEL is above 1000 mg/kg bw/day.

This subchronic toxicity study in mice is acceptable and satisfies the guideline requirement for a subchronic oral study (U.S. EPA 560/6-82-001).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 March - 19 April 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: EPA 560/6-82-001

Deviations:

yes

Remarks:

recorded observ. omitted 2 times; daily observ. animal 5 were lost due to computer malfunction; due to an oversight the necropsy quality control animals were not subm. until day 5 of the quarantine period; no signif. effect on study outcome

Principles of method if other than guideline:

duration 14 days

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP study number 84040

Limit test:

no

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

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: 173 - 304 g
- Fasting period before study:
- Housing: individually in clear, polycarbonate shoe boxes in drawer rack cages, a cellulose fiber was used as bedding; shoe boxes and bedding were changed twice weekly
- Diet, ad libitum: Certified Purina Rodent Chow 5002
- Water: 16 ounce water bottles with stoppers and sipper tubes
- Both feed and water consumption were measured weekly
- Acclimation period: 14 or 16 days (males and females, respectively) prior to study initiation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.9 - 26.7 °C
- Relative humidity (%): 29 - 43%
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Premix consisting of 50 mg nitroguanidine/kg of Rodent Chow
- Since the compound tends to clump, it was further ground in a jar mill using porcelain grindling pellets for 2 h to break up the clumps
- Nitroguanidine was then mixed into the meal in a series of 2-, 4-, and 6-fold dilutions
- Each dilution was mixed for 15 min in the jar mill
- Dilutions were then sieved through a 10-mesh screen to ensure the grinding was complete and to remove the grinding pellets

- After diet change and calculation of new diet concentrations, the appropriate amounts of premix and meal were blended together using a Model A200D mixer for at least 15 min
- Nitroguanidine was mixed into the feed at a level that, based on the feed consumption of the previous week and the animals weight, would provide the desired dose (mg/kg) on a daily basis
- All diet mixes were within 6.5% of target concentration and were adequately homogenous

- Seperate diets were prepared for males and females due to differences in food consumption and body weights due to growth

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples of the feed mixtures were analyzed to determine concentration, homogeneity, and stability of nitroguanidine in the mixtures
- Method: HPLC with diode array detector, Brownlee RP-18 column (4.6 x 250 mm), methylnitroguanidine as internal standard; external calibration with nitroguanidine and methylnitroguanidine

Duration of treatment / exposure:

14 days

Frequency of treatment:

ad libitum

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

316 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in diet

No. of animals per sex per dose:

10 animals per sex and dose

Control animals:

yes, concurrent no treatment

other: Five males and five females were used for necropsy quality controls or baseline control animals to ensure the animals were healthy and within normal limits for all measurements at receipt and after quarantine.

Details on study design:

- Dose selection rationale: based on the results of an acute toxicity study (-> acute oral median lethal dose exceeded a LIMIT dose of 5000 mg/kg -> upper dose level for pilot study) and a 14-day pilot study (no deaths nor obvious toxicity at 1000 mg/kg)
- Animal assignment: computer-based, stratified, weight-based randomization method (LAIR SOP-OP-STX-78)
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Positive control:

No positive control

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 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 / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


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 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 1 were examined.


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

Other examinations:

Clinical chemistry

Statistics:

- Animal weights, the results from haematology and blood chemistry were analyzed statistically with packaged programs available on BMDP software
- Levene's Test was used to test the equality of the variances of the groups
- Equal variances -> control group and dose groups were compared by ANOVA ; ortherwise Welch one-way ANOVA was performed
- If the F-statistic was significant in either case, the Dunnett's test was performed to determine wether or not control group was significantly different from any of the dose groups
- Total bilirubin values are non-parametric data and were analyzed using the Kruskal-Wallis one-way ANOVA

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:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- No deaths occurred during the study
- No clinical signs attributable to nitroguanidine administration were observed

BODY WEIGHT AND WEIGHT GAIN
- Weight differences due to individual growth rates, not test compound relates

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- No significant differences between control group, dose groups and sexes

WATER CONSUMPTION
- Water consumption increased in a dose-related manner during both weeks for males and females
- For the 316 and 1000 mg/kg/d dose groups this increased water consumption was significant

HAEMATOLOGY
- For the males, no significant differences in any of the haematological measurements were found
- For the females, mean cell volume was decreased in the 100 and 1000 mg/kg/d dose groups; analyses of variance indicated significant differences in the haemoglobin values, but there were no significant differences between control and treatment groups when the Dunnett's test was applied; total numbers of eosinophils, neutrophils, and monocytes were significantly lower in the 100 mg/kg/d dose group than in the control group; the values of 316 and 1000 mg/kg/d dose groups for neutrophils and the 1000 mg/kg/d dose groups for neutrophils and the 1000 mg/kg/d dose groups for eosinphils and monocytes were also lower, although not significantly

CLINICAL CHEMISTRY
- The effect of nitroguanidine on the level of several serum electrolytes, various serum biochemistry measurements, and the activity of sveral aerum enzymes was examined
- For the females, comparing the control and treatment groups by ANOVA indicated significant differences in the levels of aspartate aminotransferase, potassium, magnesium, and uric acid; however, when the Dunnett's test was performed, there were no significant differences between the control and treatment groups
- For the males, comparing the control and treatment by ANOVA and Dunnett's test indicated significant differences were present inpotassium and calcium levels ; for CA, the group means were lower for all dose groups; however, only the 100 mg/kg/d dose group was significantly lower; for potassium, there was no apparent trend with only the high-dose group being signficantly lower

ORGAN WEIGHTS
- Organ weight, organ-to-body weight ratio, and organ-to-brain weight ratios were compared for liver, spleen, adrenal, kidneys, heart, testes/ovaries, and brain weights
- For the males, the 316 mg/kg/d dose group's heart weight and heart-to-brain weight were significantly higher by both one-way ANOVA and Dunnett's test; the heart-to-body weight ratio data were significantly different by one-way ANOVA but were not significantly different by Dunnett's test
- For the females, heart weight (1000 mg/kg/d dose group significant) and heart-to-brain weight ratio (316 and 1000 mg/kg/d dose groups significant) both exhibited a decreasing trend; the brain-to-body weightt ratio exhibited an increasing trend with the 1000 mg/kg/d dose group being significantly elevated; for the 100 mg/kg/d dose group, the ovaries-to-body weight ratio was significantly elevated; the ovaries-to-brain weight ratio was also higher by one-way ANOVA but the diufference was not significant by the Dunnett's test

GROSS PATHOLOGY
- There were no microscopic lesions that could be attributed to the test compound

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

Key result

Critical effects observed:

not specified

Conclusions:

Nitroguanidine fed at dose levels from 100 mg/kg/d to 1000 mg/kg/d in the diet for 14 days was nontoxic to Sprague-Dawley rats.

Executive summary:

The 14-day subacute oral toxicity of nitroguanidine was evaluated in male and female rats. Nitroguanidine was administered in the diet at dose levels of 0, 100, 316, and 1000 mg/kg/day for 14 days. The addition of nitroguanidine to the diet did not have an effect on food consumption, but there was a significant dose-response increase in water consumption. Clinical signs attributable to the test compound were not observed during the study. At necropsy, blood samples were taken for haematological and serum clinical analyses. Serum potassium and calcium values were decreased in the treated dose groups. Microscopic examination of tissues from the control and 1000 mg/kg/day dose 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 14 days. These findings of serum electrolyte decreases coupled with increased water consumption suggest that nitroguanidine, which is excreted unchanged in the rat’s urine, may be acting as an osmotic diuretic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

good quality based on guideline studies

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Repeated dose oral toxicity of nitroguanidine was evaluated in male and female rats and mice. Nitroguanidine was administered in the diet at dose levels of 0, 100, 316, and 1000 mg/kg/day for 14 and 90 days. 

No compound related effects on mortality, clinical signs, body weight, hematology, or gross and histologic pathology were observed. Food consumption was consistently reduced in the 90 day study in rats. Causing significantly (p <= 0.05) reduced weight gains in the female high-dose group for 5 of the 13 weeks of the study period. Several serum chemistry parameters did exhibit statistically significant (p<= 0.05) alterations from the control values in the 90 day study in mice, but these changes were isolated occurrences with no consistent dose-dependency. With the exception of the brain-to-body weight ratio in the high-dose males in the 90 day study in mice, organ weights and their respective ratios were not significantly affected by dosing.

These findings indicate that nitroguanidine is nontoxic in rats and mice when administered at doses as high as 1000 mg/kg/day for up to 90 days.

The findings of increased water consumption suggest that nitroguanidine, which is excreted unchanged in the urine, may be acting as an osmotic diuretic.

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

Justification for classification or non-classification

No compound related effect could be established in subchronic oral toxicity studies in rodents up to 1000 mg/kg bw/day. Nitroguanidine is considered non- toxic with respect to repeated dose toxicity.