1-nitroguanidine

Endpoint summary

Description of key information

Two reliable acute toxicity studies are available. An oral study in mice with an MLD in female mice of 4345 mg/kg bw

and a dermal study in rabbits indicating no acute effect up to 2000 mg/kg bw.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

Test substance supplier not specified

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

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

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).

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

4 345 mg/kg bw

95% CL:

> 3 483 - < 5 335

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

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

4 345 mg/kg bw

Quality of whole database:

good quality based on guideline study

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Sep - 10 Oct 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA Office of Pesticides and Toxic Substances (Ts-792); Acute exposure, dermal toxicity. In: Health Effects test guidelines. Washington DC; 1982; EPA 560/6-82-001

Deviations:

yes

Remarks:

The animals were not submitted to necropsy until 15 days after dosing. Animals were observed on the 15th day before being submitted for necropsy. The food for the animals was removed early on the 14th day and was returned later that morning after the next

Principles of method if other than guideline:

according to: Acute dermal toxicity study. LAIR Standard Operation Procedure OP-STX-30, Letterman Army Institute of REsearch, Presidio of San Francisco. 1984

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP study number 84025

Test type:

fixed dose procedure

Limit test:

yes

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, Watsonville, CA 95076
- Age at study initiation: young
- Weight at study initiation: 2539 - 3900 g
- Housing: individually in stainless steel, wire mosh bottom, battery-type cages with automatic flushing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 19 days (quarantained)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20 C°
- Humidity (%): 50-70 % (spikes up to 85% during room cleaning)
- Photoperiod (hrs dark/hrs light): 12 h/12h

IN-LIFE DATES: From: 25. Sept 84 To: 10. Oct 84

Type of coverage:

occlusive

Vehicle:

physiological saline

Details on dermal exposure:

TEST SITE
- Area of exposure: 300 cm^2
- Type of wrap if used: gauze + hypoallergic tape (Durapore Surgical tape, 3M Corp) + Vetrap bandaging tape (Animal Care products, 3M Corp)


REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiped with gauze moistured with water
- Time after start of exposure: 24 h


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 g/kg
- Concentration (if solution): 0.25 g per ml salino
- Constant volume or concentration used: yes

Duration of exposure:

24 h

Doses:

2 g/kg

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 1, 2, 4 h after dosing and daily for the remainder of the study. A second "walk adjathrough" was performed daily with only significant observations recorded. Dermal observations also were recorded daily according to severity, area, and location.
- Necropsy of survivors performed: yes

Statistics:

not applicable

Preliminary study:

not applicable

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

none

Clinical signs:

other: none

Gross pathology:

Female:
84F584 - "[..] lymph nodes were greatly enlarged (up to 9 cm diameter), white and lobular. The nodes contained [..] white creamy material."
84F585 - no lesions recognized.
84F586 - no lesions recognized.
84F587 - no lesions recognized.
84F588 - The skin had a small (2 x 8 cm) area of slight erythema in the midlumber area.

Male:
84F591 - The skin had a small (2 x 3 cm) area of slight erythema in the midlumber area.
84F592 - There was a purulent, bilateral otitis media.
84F593 - The skin had a small (5 x 7 cm) area of slight erythema in the midline, dorsal thoracic area. The kidneys had bilateral tan mottled areas over the entire cortex.
84F594 - no lesions recognized.
84F595 - There was a purulent, bilateral otitis media.

- Both dosed and control skin section contain minimal, small aggregates of macrophages, heterophils and lymphocytes in the superficial dermis and adjacent to hair follicles. Dosed and control skin sections have a few follicles with individual or small groups (3 -6 cells) of necrotic cells in the external root sheath. There was no difference in severity of skin lesions between dosed and control skin sections.

- No compound related lesions and/or deaths were seen. Otitis media was considered an incidential finding.

- Tan, mottled, bilateral areas over the cortex of the kidney may have been caused by Encephalitozoon cuniculi, a commonprotozoen parasite of rabbit kidney.

- The erythema observed grossly in rabbits 84F588, 84F591 and 84F593 could not be directed microscopically and most likely was discoloration on the skin surface rather than erythema.

Interpretation of results:

GHS criteria not met

Conclusions:

A limit dose of 2 g/kg of nitroguanidine was not lethal following dermal exposure for 24 hours, and produced no compound-related clinical signs or dermal irritation during the 15 day observation period. Nitroguanidine possesses a minimal potential for acute dermal toxicity.

Executive summary:

In an acute dermal toxicity study according to GLP, groups of New Zealand White Rabbits (5/male, 5/female) were dermally exposed to nitroguanidine (99.4 %) in physiol. saline for 24 h to approximately 300 cm² at a LIMIT dose of 2 mg/kg bw. Animals then were observed for 15 days.

There were no treatment related deaths, clinical signs or necropsy findings. Nitroguanidine possesses a minimal potential for acute dermal toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

The MLD values for nitroguanidine in ICR mice were 4345 mg/kg for females and above 5620 mg/kg for males. Gastro-intestinal tract, urogenital system, and central nervous system were considered to be target organs for nitroguanidine .

A limit dose of 2 g/kg of nitroguanidine was not lethal following dermal exposure of New Zealand White Rabbits for 24 hours, and produced no compound-related clinical signs or dermal irritation during the 15 day observation period.

Toxicity by inhalation could not be assessed. It was technically not feasable to produce an aerosol that would be concentrated high enough to assess high dose effects.

Justification for classification or non-classification

According Regulation (EC) No 1272/2008 LD50-doses of more than 2000 mg/kg are not considered to indicate oral or dermal acute toxicity.