1-nitroguanidine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1985 - March 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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-40; Developmental Toxicity Study

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

dated from February 8th 1988

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin-Kingman, Fremont, California
- Age at study initiation: Phase I: males 82 days, females 112 days; Phase II: males 97 days, females 95 days
- Weight at study initiation: Phase I: males: 312-444 g, females 259-332 g; Phase II: males 389-468 g, females 209-290 g
- Housing: individually in wiremesh rack cages, pregnant females in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 75.1 +-2.0
- Humidity (%): 45.0 +- 7.0
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

CMC (carboxymethyl cellulose ) sodium salt, high viscosity

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Initially, a smooth paste containing nitroguanidine and a small amount of vehicle was prepared in a mortar with a pestle. Vehicle was then added gradually until the final volume was obtained. The concentrations prepared were 20 mg/ml for the 100 mg/kg/day dose, 63.2 mg/ml for the 316 mg/ml/day dose, and 200 mg/ml for the 1000 mg/kg/day dose. The dosing suspensions and vehicle control were given at a volume of 5 ml/kg body weight. The vehicle and dosing suspensions were prepared prior to the start of dosing for each study phase and refrigerated. Before the animals were dosed each day, the containers of dosing preparation were placed in a beaker of hot tap water for 15 and 30 minutes to bring the suspensions to room temperature. Chemical analyses for accuracy and homogeneity of the dosing suspensions were conducted.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: Nitroguanidine is not soluble in water at the concentrations tested. Carboxymethylcellulose holds nitroguanidine in a homogeneous suspension.
- Amount of vehicle (if gavage): 1%-solution
- Source: Sigma Chemical Co. , St. Louis, MO

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogenecity
A suspension of nitroguanidine (200 mg/ml, 300 ml) was prepared in 1% carboxymethylcellulose. This suspension was subsequently used to prepare two more dilute suspensions of approximately 60 mg/ml (20 ml) and 20 mg/ml (20 ml) in 20-ml vials. The suspensions were stirred well, and aliquots of 1 ml were removed from the top, middle, and bottom layers of each suspension. The aliquots were transferred to either 500 or 1000 ml volumetric flasks and diluted to volume with water. After one more dilution the optical absorbance at 264 nm was determined.
The concentration of the original suspension was then calculated using the dilution and absorbance data. A comparison of the individual values to the mean value of the appropriate group showed no deviation larger than 3%.

Chemical Analysis
All dosing suspensions were analyzed by transferring 1 ml aliquots of suspension to a volumetric flask and diluting to volume. An aliquot of the first dilution was subsequently transferred to a second volumetric flask and diluted to volume. The absorbance spectrum (200-340 nm) of the final dilution was determined with a UV/VIS spectrometer. The absorbance at 260 nm was then used to calculate the concentration of nitroguanidine according to an equation based on Beer’s law.

Details on mating procedure:

After the quarantine period, each male was placed in breeding cage with two females. Females were checked each morning for evidence of insemination. Day 0 for each female was the day sperm were observed in her vaginal smear. Spermpositive females were separated from the males and caged individually. Those females which were not sperm positive at the completion of the breeding period were removed from the study.

Duration of treatment / exposure:

10 days (from day 6 through day 15)

Frequency of treatment:

daily

Duration of test:

Phase I: 21 Oct 85 through 9 Nov 85
Phase II: 24 Feb 86 through 13 Mar 86

No. of animals per sex per dose:

27 females 0 mg/kg/day
27 females 100 mg/kg/day
23 females 316 mg/kg/day
27 females 1000 mg/kg/day

Control animals:

yes

Details on study design:

- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Daily from day 0 - 20

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 10, 15, and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Visceral examination

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

Fetuses were assigned alternatively to either skeltal or visceral examination
- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:

Statistics:

Data from both phases were combined for analysis. The litter or litter mean was used as the experimental unit. All tests were run at the 0.05 level of significance. The maternal body weights, weight changes, food consumption, and fetal weights and lengths were compared by one-way analysis of variance. Then, if a significant F value occurred, the Newman-Keuls test was applied to the data. The implementation efficiency, percent resorptions, and percent live and dead fetuses, and ossification were compared by the one-way Kruskal-Wallis test. If the Kruskal-Wallis test was significant, the Mann-Whitney test was used to determine which groups were different. The fetal examination findings were compared by chi-square analysis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Nitroguanidine did not affect the pregnancy rate.
Seven animals died during the study, and one moribund animal was terminated. Five of the seven animals died as a result of difficulties administering the concentrated dosing suspension.
When given at 1000 mg/kg/day, nitroguanidine produced weight loss during the treatment period, Days 6 to 15, and decreased weight gain during the study period, day 0 to corrected day 20, in comparison to the control. Food consumption also was decreased significantly during the treatment period in the 1000 mg/kg/day dose group. Lower doses of nitroguanidine did not adversely affect maternal weight gain or food consumption.
Clinical signs, which occurred with a high frequency in the 1000 mg/kg/day group during the treatment period included red urine, dehydration, red material on nose/whiskers, red material on forelimbs, and hunched posture. Clinical signs occurred in 100% of the 1000 mg/kg/day group versus 39% of the control group during the treatment period and in 29% of the 1000 mg/kg/day group animals compared with 9% in the control group during the posttreatment period.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Nitroguanidine had no effect on the number of corpora lutea, implantations, resorptions and alive and dead fetuses.
Nitroguanidine did not affect the male-to-female ratio. Male and female fetuses from the 1000 mg/kg/day dose group were significantly lighter in weight and shorter in length than the controls. There was no size difference in the 100 and 316 mg/kg/day dose group fetuses in comparison to the controls.
A variation seen at low frequency was haematoma. Three fetuses had external malformations. One control group fetus had anasarca and abnormal body shape in which the body was short and thick, particularly through the neck. In the 316 mg/kg/day group, one fetus had bilateral anophthalmia, hypoplastic pinnae, absent lower jaw, and abnormal body shape (square) and one fetus from a different litter had anasarca. These variations and malformations are not dose-related.
Slightly dilateral renal pelvis was the most frequently observed visceral variation, occurring in all groups with the highest incidence in the control group. The occurring spontaneous malformations are not dose-related.
Two fetuses had skeletal malformations, which are considered spontaneous because they are not dose-related and occurred at a low frequency.
The fetuses from the 1000 mg/kg/day group had significantly fewer ossified metacarpals and metatarsals, but this decrease was not significant. Additionally, the fetuses from the 1000 mg/kg/day group had a higher incidence of reduced ossification of the pubis than the controls. There was a dose-related decrease in the incidence of rudimentary lumbar ribs, ranging from 17.7% in the control to 11.8 in the 100 mg/kg/day dose group.
There was no significant difference in the rate of malformations among the dose groups. The number of fetuses, but not the number of the dose litters, with skeletal variations was increased in the 1000 mg/kg/day dose group in comparison to the control.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Nitroguanidine is not been considered a selective reproductive or developmental toxicant under conditions of this study. All effects were seen at the highest dose level of 1000 mg/kg bw/day in the presence of overt maternal toxicity. Fetal toxicity could be attributed to maternal toxicity. The NOAEL for teratogenicity is 1000 mg/kg/day.

Executive summary:

In a developmental toxicity study Nitroguanidine (100%) was administered to 18–24 Sprague-Dawley rats/dose by gavage at dose levels of 0, 100, 316, or 1000 mg/kg bw/day from days 6 through 15 of gestation.

Nitroguanidine given at 1000 mg/kg/day produced decreased food consumption, weight loss, dehydration, red urine, and red material on nose/whiskers in the dams during the treatment period and decreased weight gain from Day 0 to Day 20 of gestation. The maternal LOAEL is 1000 mg/kg bw/day, based on body weight gain, food consumption and clinical signs. The maternal NOAEL is 316 mg/kg bw/day.

Fetotoxicity was seen only in the presence of overt maternal toxicity. Retarded ossification of the sternebrae, caudal vertebrae, and pubis was reported in the 1000 mg/kg bw/day group. There was no evidence of developmental toxicity. The NOAEL for fetotoxicity in this study is 316 mg/kg and can be attributed to maternal toxicity. The NOAEL for developmental toxicity is 1000 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (similar to OECD 414) in rat.