1-nitroguanidine

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Sep -7 Nov 1984

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

Qualifier:

according to guideline

Guideline:

other: LAIR Standard Operating Procedure OP-STX-36, "Acute Oral Toxicity Study" and EPA guidelines

Deviations:

yes

Remarks:

see below (overall remarks): None of the changes/deviations is believed to have had an adverse effect on the study

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP study number 84009

Test type:

acute toxic class method

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 not specified

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc.
- Age at study initiation: approx. 3 months
- Weight at study initiation: 21 - 40 g
- Fasting period before study: no data
- Housing: individuals in stainless stell, wire-mesh cages in racks equipped with automatically flushing dumptanks; no bedding
- Diet, ad libitum: Certified Purina Rodent Chow Diet 5002
- Water: continous drip from central line
- Quarantine/Acclimation period: 20 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3-26.1
- Humidity (%): 34-68
- Photoperiod (hrs dark/hrs light): 12/12


IN-LIFE DATES: From: 26 Sep 1984 To: 7 Nov 1984

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Nitroguanidine was prepared as a suspension in 0.2 % methylcellulose and 0.4 % Tween 80 in sterile water

Details on oral exposure:

VEHICLE
- Nitroguanidine was prepared as a suspension in 0.2 % methylcellulose and 0.4 % Tween 80 in sterile water
- Methylcellulose (4000 grade): Lot 82F-0634, expiration date April 1994, source: Sigma Chemical Co (St Louis, MO)
- Tween 80 (polyethoxylene (20) sorbitan monooleate): Lot 713137, exp. date December 1986, source: Fisher Scientific Products (Fairlawn, NJ)
- Sterile water for injection: Lot 49-420-DM-03, exp. date 1 February 1985, source: Abbott Laboratories (Chicago, IL)
- Concentration in vehicle:
- Amount of vehicle (if gavage): the volume of the dosing suspension that each animal received was based upon its assigned dosage level, its body weight and the nitroguanidine concentration of the suspension; to keep the volume administered at any time below 10 ml/kg, all nitroguanidine groups received split dosings (2 separate gavages), administered within 90 minutes of each other; half of the vehicle control animals also received split dosings while the other half received a single gevage; for any single gavage, the volume administered renaged from 0.28 to 0.40 ml in the males and from 0.14 to 0.32 in the females
- Justification for choice of vehicle: at the concentrations necessary to achieve the dosage levels set for this study, nitroguanidine is insoluble in water or saline; dosing was therefore performed with a highly concentrated suspension of nitroguanidine.



MAXIMUM DOSE VOLUME APPLIED:
10 ml/kg

Doses:

- Males: 5620 mg/kg + vehicle control group
- Females: 6310, 5010, 3980, 2820 mg/kg + vehicle control group

No. of animals per sex per dose:

- 10 animals per dose
- Males: one dose group + vehicle control group
- Females: four dose groups + vehicle control group

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: daily
- Frequency of weighing: weekly
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

- For the females, the LD10, LD50, and LD90 were derived by probit analysis using the maximum likelihood method described by Finney
- the program, PROBIT, developed for the Data General Computer, Model MV8000, was used to determine the probit curve and lethal dose value

Lethal Dose Calulations:
- Lethal dose values for the females were calculated by probit analysis, and the equation for the probit regression line was: Y = -16.4 + 5.89 Log X, where X is the dose and Y the corresponding probit value
- Misdosed animals were excluded from statistical analysis and eliminated from the study

Results and discussion

Preliminary study:

A pilot study, performed with a dose above the "LIMIT" value of 5000 mg/kg , was conducted. Results for the males were equivocal, and the Limit test was repeated during the test phase. The test phase for the males consisted of a treated group, which received a dose (5620 mg/kg) that was in excess of the LIMIT test dose, and a vehicle control group. For the females, preliminary results suggested an approximate lethal dose slightly lower than the LIMIT value of 5000 mg/kg. Dose levels for the test phase were set accordingly (6310, 5010, 3980, 2820 mg/kg, and vehicle control).

Effect levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

4 345 mg/kg bw

95% CL:

> 3 483 - < 5 335

Effect levels 2

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

5 620 mg/kg bw

Mortality:

- Mortality in the male LIMIT group (5620 mg/kg) was 40%; 4 of the 10 animals died; two of these deaths occurred within 24 hours of dosing, one within 48 hours, and one 11 days after dosing
- Of the 40 females administered doses ranging from 2820 to 6310 mg/kg, 20 died during the administration period; eleven (55%) of the deaths occurred during the initial 24 hours after dosing and 8 more died between 24 and 50 hours. Thus, 19 of the 20 (95%) deaths occurred within 50 hours of dosing; the remainig death occurred within 75 hours after dosing
- Although nitroguanidine was more potent in female mice, the mechanism of its toxic action appeared to be similar to its action in male mice as most of the deaths in both sexes occurred during the 48 hours following administration of nitroguanidine.

Clinical signs:

other: - Primarily referable to gastrointestinal (GI) tract and the central nervous system (CNS); also urogenital system - Most common signs: hunched posture and inactivity; occurred together often - Since the males responded with less than 50% mortality to a d

Gross pathology:

- Few gross lesions were observed and none of these appeared related to administration of nitroguanidine
- At necropsy, none of the animals succumbing to nitroguanidine, nor the survivors, exhibited any pathology of the GI tract
- 2 of 4 males succumbing to nitroguanidine exhibited urinary bladder distention; penile paraphimosis was observed in one of these 2
- The other 2 males dying acutely and five of the six survivors were not remarkable at necropsy
- One of the surviving males, which had developed ocular opacities, exhibited cataract formation in the one lens examined microscopically

- One of the 20 females dying acutely after administration of nitroguanidine (at doses 2820 to 6310 mg/kg) exhibited any gross lesions attributable to nitroguanidine
- One female survivor (3980 mg/kg) presented at study termination with focal lesions in the right salivary gland and the liver; the salivary gland was not remarkable upon microscopic examination; foci of coagulative necrosis in the liver were observed during microscopic examiniation; these areas did not respond to special bacterial stains and may have been due to mouse hepatitis virus;
- Microscopic examinations of the eyes from one control female (vehicle only) revealed loss or reduction of retinal rod and cone layers
- All the other females surviving until study termination were not remarkable at necropsy.

Other findings:

- Following dosing, a white crystalline material was observed in the urine of many of the animals, male and female, receiving nitroguanidine
- This material is presumed to be nitroguanidine because in a related study, rats dosed with nitroguanidine excreted a similar white crystalline material which was identified by HPLC as nitroguanidine

Any other information on results incl. tables

Table 1: Calculated Lethal Doses (LD) of Nitroguanidine

Effect Level	Dose (mg/kg)	95 % Confidence Limits (mg/kg)
FEMALES
LD10	2632	(1108; 3341)
LD50	4345	(3483; 5335)
LD90	7171	(5693; 16378)

Because less than 50% mortality was produced by a dose (5620 mg/kg) in excess of the LIMIT test dose, dose-response data were not obtained for the males.

Table 2: Incidence Summary for Clinical Observations in Male Mice Dosed With Nitroguanidine

Clinical Signs	Group Dose (mg/kg) (n=)	Limit 5620 10	Vehicle Control 10
Hunched Posture		8	0
Inactivity		7	0
Rough Coat		3	0
White Crystalline Material in Urine		4	0
Yellow Perianal Staining		3	0

 

Table 3: Incidence Summary for Clinical Observations in Female Mice Dosed With Nitroguanidine

Clinical Signs	Group Dose (mg/kg) (n=)	5F 2820   10	3F 3980   10	2F 5010   10	1F 6310   10	4F Vehicle   9
Hunched Posture		6	7	5	9	0
Inactivity		4	5	6	8	0
Seizure		2	4	4	9	0
White Crystalline Material in Urine		9	3	3	7	0
Irritability		1	3	0	1	0
Hyperactivity		0	3	0	0	0

 

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The MLD values for nitroguanidine in ICR mice were 4345 mg/kg bw for the females and above 5620 mg/kg bw for the males. This places nitroguanidine in the "practically nontoxic" to "slightly toxic" classification. Target organ systems for nitroguanidine were the gastro-intestinal tract, the urogenital system, and the central nervous system.

Executive summary:

The acute oral toxicity of nitroguanidine was determined in male and female Institute of Cancer Research (ICR) mice using the oral gavage method. Groups 10 animals per concentration and vehicle control were dosed at 5620 and 0 (vehicle control) mg/kg (males) and at 6310, 5010, 3980, 2820, and 0 (vehicle control) mg/kg (females). Nitroguanidine was prepared as a suspension in 0.2 % methylcellulose and 0.4 % Tween 80 in sterile water. Animals were then observed for 14 days.

 

Clinical signs produced by nitroguanidine were consistent with general malaise, effects on the gastrointestinal (GI) tract and urogenital system (CNS). A frequent observation was the presence of a whitish crystalline material in the urine. In a previous study, chemical analyses of a whitish crystalline material isolated from the urinary bladder of a rat administered nitroguanidine orally indicated that the crystalline material was nitroguanidine.

In males, tests at or above the LIMIT value of 5000 mg/kg produced less than 50 % mortality. In females, the median lethal dose was 4345 (95% C.I.: 3483-5335) mg/kg.

Based on the results of this study, nitroguanidine is placed in the "practically nontoxic" to "slightly toxic" classification. Target organ systems for nitroguanidine were the gastro-intestinal tract, the urogenital system, and the central nervous system.

 

 

This acute oral study is classified as acceptable. It does satisfy the requirement for an acute oral study in the mice.