1-nitroguanidine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

no adverse effect observed

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1987 - May 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: LAIR SOP, OP-STX-79 "Reproductive and Fertility Toxicity Study"

Deviations:

yes

Remarks:

(deviations (dose levels different from those specified in the protocol; changes in the animal identification methods) did not influence the overall results of the study)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

(no details on sperm morphology and motility)

GLP compliance:

yes

Remarks:

GLP Study Number 87012

Limit test:

no

Specific details on test material used for the study:

Test substance supplier Sunflower AAP
Assey 99.7%

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: (P) 8 wks
- Weight at study initiation: (P) Males: 217 - 272 g; Females: 166 - 240 g
- Fasting period before study:
- Housing: * Individually in clear, polycarbonate shoe boxes in drawer reck cages;
* During breeding females were placed in the the males' cages;
* Pups were housed with their dams until weaned at 21 days of age
* Alpha-dri beeding (cellulose fibre from Shepherd Specialty Papers, Kalamazoo, MI)
* Boxes and bedding were changed weekly or sooner if wet or dirty
- Diet: ad libitum; Purina Certified Rodent Chow 5002 Meal Form
- Water: ad libitum; water purified by reverse osmosis was provided by automatic water dispensers
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): * 16-20 °C during acclimation period
* 20-27 °C thereafter with occasional spikes to 28 °C
- Humidity (%): 35-54 (occasional spikes as high as 86% and as low as 21 %)
- Air changes (per h):
- Photoperiod (hrs dark/hrs light): 12/12 with 0.5 h dawn phase in and 0.25 h dusk phase out

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): at least every three weeks
- Mixing appropriate amounts with (Type of food): Rodent Chow

Details on mating procedure:

- M/F ratio per cage: 1/1 (same dose level)
- Length of cohabitation: * Until insemination occurred or one week had elapsed;
* If mating not occurred after one week, the male was replaced
* If mating had not occurred during the second week, the second male was replaced with a third male for one week
* Females that had not been bred after 21 days were sent to necropsy
- Proof of pregnancy: sperm in vaginal smear referred to as "gestation day 0"
- This breeding procedure was followed for the parental and F1 generations
- Breeding began for the parental-generation animals when they were 18 weeks old and had been receiving the nitroguanidine diet for 10 weeks
- Breeding began for the F1-generation animals when they were approx. 18 weeks old; F1-generation siblings were not mated
- The day of delivery was designated "lactation day 0" for each female
- The F1-pups selected to continue in the study were housed individually on lactation day 21 (weaning)

Mating: weight-based stratified randomization LAIR SOP OP-STX-78

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Verification of concentrations after mixing of test substance into food
- All nitroguandine diet mixtures were within 10.0% of the target concentration and were homogenous

Duration of treatment / exposure:

The diet was fed to the parental males and females starting at 56 to 58 days of age and continued throughout their lives and to the F1 and F2 generation animals (until scheduled necropsy).

Frequency of treatment:

continous treatment

Details on study schedule:

- F1 parental animals were not mated until 18 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Remarks:

Doses / Concentrations:
0, 1.3, 4.0, and 12.7 ppt
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 100, 316, 1000 mg/kg bw
Basis:
nominal in diet
based on bw of young adult rats

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

- Food consumption was measured weekly except during cohibation for breeding

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily throughout the study
- During the study periods when litters were being delivered, "walk through" observations were made frequently throughout the day
- Females that were found delivering litters were recorded and observed for behavioral changes and signs of difficult or prolonged delivery
- Cage side observations checked in table were included.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes
- During breeding feeders of the paired animals were not weighed
- Food consumption was not monitored for periods less than one week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
- Litters of eight or fewer were not adjusted
- Runts were not culled; if a pup randomly selected to continue the study was not progressing well and seemed unlikely to survive to weaning, it was culled


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
- each litter was observed as soon as possible after delivery
- the dam was weighed
- each live pup was examined for external appearance, sexed, weighed, and marked with an identifying code
- dead pups were examined for external appearance and preserves in Bouin's solution for subsequent visceral examination
- during the lactation period (lactation day 0 to weaning on lactation day 21), litters were examined daily; each pup was accounted for; dead, missing, and partially cannabilzed pups were recorded
- pups found dead were fixed in Bouin's solution for subsequent visceral examination
- the dam and each pup were weighed individually on lactation days 0, 4, 7, 14, and 21

Postmortem examinations (parental animals):

SACRIFICE
- Parental animals were euthanized at the end of breeding
- Females that did not breed after being housed with a male for 21 days and bred females that did not give birth by gestation day 24 were euthanized
- dams were euthanized after lactation day 21



GROSS NECROPSY
- A complete gross examination, with special attention to the organs of the reproductive system, was performed on all adult animals
- The following organs and tissues from all parental animals selected for mating were preserved: vagina, uterus, ovaries, testes, epididymus, seminal vesicles, prostate, and pituitary gland


HISTOPATHOLOGY/ORGAN WEIGHTS
- Histopathological examination was performed on the high dose and the control group animals

Postmortem examinations (offspring):

SACRIFICE
- F1 were euthanized at the end of breeding
- Females that did not breed after being housed with a male for 21 days and bred females that did not give birth by gestation day 24 were euthanized
- Dams were euthanized after lactation day 21
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after lactation day 21.
- Pups found dead and pups culled on lactation day 4 were fixed in Bouin's solution and subsequently examined under a dissecting microscope by the modified Wilson freehand razor blade sectioning technique


GROSS NECROPSY
- A complete gross examination, with special attention to the organs of the reproductive system, was performed on all adult animals
- The following organs and tissues from all F1 animals selected for mating were preserved: vagina, uterus, ovaries, testes, epididymus, seminal vesicles, prostate, and pituitary gland
- A gross examination was performed on the weanling animals


HISTOPATHOLOGY / ORGAN WEIGTHS
- Histopathological examination was performed on the high dose and the control group animals

Statistics:

The body weights, food consumption, and reproductive data were processed by reproductive/teratology software. Body weight, food consumption, pup and litter weights, length of gestation, number of pups delivered, and pup per litter were compared by one-way analysis of variance and Dunnett’s test. The male and female reproductive indices, survival, and sex ratio were compared by Fisher’s Exact 2-tailed test. Test were run at the 0.05 and 0.01 level of significance and compared each dose group with the control group. Clinical observations and examinations on the pups at birth and after processing in Bouin’s solution were listed for individual animals and tabulated by group.
Pathology data was processed by Xybion pathology software, Cedar Knolls, NJ.

Reproductive indices:

Female mating index, female fertility index and gestation index were determined (P & F1)
Effects on male reproduction were examined by means of bred females, bred females pregnant, sired litters and average days to mate (P & F1)

Offspring viability indices:

Live birth index; Pups surviving 4 days viability index and pups surviving 21 days lactation index were determined (P & F1)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL AND F1 ANIMALS)
- One adult animal, an F1 male from the low dose group, died during the study; the cause of death was not determined by necropsy.
- Red stains, particularly on the nose, and hair loss from the limbs occurred frequently in all dose groups, both sexes, and both generations
- None of the clinical signs were dose related
- Both males and females of all dose groups in the F1 generation had a higher incidence of irritability than the parental animals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL AND F1 ANIMALS)
- No dose-related differences in the male or female body weights from the start of the study until mating in the parental generation
- No dose-related differences in the male or female body weights from weaning until the tenth week post-weaning in the F1 generation
- From the tenth week until mating both the males and females in the F1 high dose group were significantly lighter than the control group, initially at p < 0.05, then at p < 0.01

Maternal gestation body weights:
- The high dose group parental females were significantly lighter, p < 0.05, than the control group on gestation days 7, 14 and 21
- The low dose group group F1 females were significantly lighter, p < 0.05, than the control group on gestation day 21
- The high dose group F1 females were significantly lighter, p < 0.01, than the control group on gestation days 14 and 21

- There were no dose-related changes in the body weights of the parental generation animals.
- The mid dose F1 females were singnificantly lighter than the control group on gestation days 14 and 21.
- However, the number of animals in the "bred did not deliver" category was small and varied between groups.

Maternal postpartum body weights:
- The parental generation high dose females were significantly lighter than the control animals on lactation days 0, p < 0.05, and 14, p < 0.01
- The F1 generation high dose females were significantly lighter than the controls on days 0 and 7 at p < 0.01 and on day 14 at p < 0.05


FOOD CONSUMPTION (PARENTAL AND F1 ANIMALS)
- Significantly lower (p < 0.05) for high dose parental females than the controls for weeks 2 and 7
- F1 males: significantly lower (p < 0.05) than the controls for the low dose group on week 3 and for the high dose group on weeks 3, 4, and 10
- The high dose group F1 females food consumption was lower, p < 0.05, than the controls for week 3


REPRODUCTIVE DATA
- No dose-related differences in the mating, female fertility, gestation, or live birth indices, length of gestation, number of live pups per litter, or sex ratio in either generation

HISTOPATHOLOGY (PARENTAL ANIMALS) -> of the reproductive organs:
- No lesions attributable to nitroguanidine in any of the generations

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: lack of toxicity to reproductive or fertility

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- The low dose parental litters had significantly, p < 0.01, fewer pups surviving 4 days (viability index) and the low and mid dose group litters had significantly, p < 0.01, fewer pups surviving 21 days (lactation index) than the control litters
- The high dose group had significantly, p< 0.05, more pups dying, missing, and/or cannabilzed during lactation days 8-14 than the control litters
- However, there were no differences in the parental high dose group viability or lactation indices compared to the controls
- The mid dose group F1 generation litters had significantly , p < 0.01, more pups surviving 4 days than the control litters

CLINICAL SIGNS (OFFSPRING)
- The incidence of findings was extremly low
- Findings were varied and occurred in all dose groups

BODY WEIGHT (OFFSPRING)
- Pup weights were not affected with one reception: the male pups in the high dose group parental litters were significantly lighter than the control litters on day 7 only; since there was no significant difference in weights for these pups on any of the other weighing days (day 0 to day 21) this difference was considered an aberration.

VISCERAL EXAMINATIONS:
- Dilated renal pelvis of slight or moderate severity occurred in low frequency in all dose groups of both generations
- Dilated renal pelvis of of marked severity occurred in slightly higher frequency in the F2 generation than in the F1 generation and in the F2 generation occurred in more pups (but not more litters) in the high dose group than in the controls or lower dose groups
- Underdeveloped kidneys in which the medulla was absent, cortes thin, and cavity enlarged occurred in slightly higher frequency in the F2 generation than in the F1 generation; it was not dose related
- "Oviduct not straight" was the description used for the oviducts which were bent, twisted, zigzagged, or curled over themselves
- "Ectopic" was used to discribe a kidney or ovary which was slightly out of place in the body
- The only major malformations occurred in one pup in the low dose group in the F2 generation which was dead at the initial litter examination; visceral findings on this pup were anopthalmis, short jaw, small left atria, septum of the heart ventricles absent and tissue adema
- The other visceral findings in this study were slight variations from normal to slightly delayed development


PATHOLOGY (OFFSPRING)
- No lesions were observed which were attributable to treatment
- Lesions which were observed were generally slight to mild, and were considered to be incidental finfdings of little or no clinical significance
- Terminal body weights of the F1 females in the low and high dose groups and F1 males in the high dose group were significantly lower than the control groups

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lack of toxicity to reproductive or fertility

Key result

Reproductive effects observed:

not specified

Summary P & F1

Mortalities

One adult animal, an F1 male from the low dose group, died during the study. The cause of death was not determined by necropsy.

 

Body Weights

Individual body weights were determined for parental and F1 males and females. There were no dose-related differences in the male or female body weights from the start of the study until mating in the parental generation. There were no dose-related differences in the male or female body weights from weaning until the tenth week post-weaning in the F1 generation. From the tenth week until mating both the males and females in the F1 high dose group were significantly lighter than the control group, initially at p<0.05, then at p<0.01: Maternal gestation body weights are. The high dose group parental females were significantly lighter, p<0.05, than the control group on gestation days 7, 14 and 21. The low dose group F1 females were significantly, p<O.O5, lighter than the control group on gestation day 21. The high dose group F1 females were significantly, p<0.01, lighter than the control group on gestation days 14 and 21.

There were no dose-related differences in the body weights of the parental generation animals. The mid dose group F1 females were significantly lighter than the control group on gestation days 14 and 21. However, the number of animals in the "bred did not deliver" category was small and varied between groups. The parental generation high dose females were significantly lighter than the controls on lactation days 0, p<0.05, and 14, p<0.01. The F1 generation high dose females were significantly lighter than the controls on days 0 and 7 at p<0.01 and on day 14 at p<0.05.

 

Food Consumption

Individual weekly food consumption was recorded. Occasionally throughout the study, feeders were tipped or wet and no weekly weight was recorded for that animal. Food consumption of the high dose parental females was significantly lower, p<0.05, than the controls for weeks 2 and 7. Food consumption of the F1 males was significantly lower, p<0.05, than the controls for the low dose group on week 3 and for the high dose group on weeks 3, 4, and 10. The high dose group F1 female food consumption was lower, p<0.05, than the controls for week 3.

 

Clinical Signs

Individual clinical signs are reported. Red stains, particularly on the nose, and hair loss from limbs occurred frequently in all dose groups, both sexes, and both generations. None of the clinical signs were dose related. Both males and females of all dose groups in the F1 generation had a higher incidence of irritability than the parental animals.

 

Reproductive Data

There were no dose-related differences in male breeding data.

Individual delivery and litter data, the number of pups born live and dead, the number of pups surviving on days 0, 4, 7, 14 , and 21, and the length of gestation, are recorded for the parental and F1 generation litters, respectively. The individual litter pup sex and post-partum status are summarized for the parental and F1 generation litters, respectively, as well as summaries of reproductive, delivery, and litter data by dose groups. There were no dose-related differences in the mating, female fertility, gestation, or live birth indices, length of gestation, number of live pups per litter, or sex ratio in either generation. The low dose parental litters had significantly, p<0.01, fewer pups surviving 4 days (viability index) and the low and mid dose group litters had significantly, p<0.01, fewer pups surviving 21 days (lactation index) than the control litters. The high dose group had significantly, p<0.05, more pups dying, missing, and/or cannibalized during lactation days 8-14 than the control litters. However, there were no differences in the parental high dose group viability or lactation indices compared to the controls. The mid dose group F1 generation litters had significantly, p<0.01, more pups surviving 4 days than the control litters.

 

Pup Body Weights

Individual pup body weights and litter means for lactations days 0, 4, 7, 14, and 21 are recorded for the parental and F1 generation litters, respectively. Nitroguanidine administration did not affect the pup weights, with one exception. The male pups in the high dose group parental litters were significantly lighter than the control litters on day 7 only. Since there was no significant difference in weights for these pups on any of the other weighing days (day 0 to day 21) this difference was considered an aberration.

 

Examination of Pups

The findings from examination of pups soon after delivery are presented recorded for the F1 and F2 generations, respectively. The incidence of findings was extremely low. Findings were varied and occurred in all dose groups. Pups that were found dead throughout the 21 day lactation period and pups that were culled on day 4 were preserved in Bouin's solution for subsequent visceral examination. Dilated renal pelvis of slight or moderate severity occurred in low frequency in all dose groups of both generations. Dilated renal pelvis of marked severity occurred in slightly higher frequency in the F2 generation than in the F1 generation and in the F2 generation occurred in more pups (but not more litters) in the high dose group than in the controls or lower dose groups. Underdeveloped kidneys in which the medulla was absent, cortex thin, and cavity enlarged occurred in slightly higher frequency in the F2 generation than in the F1 generation. The only major malformations occurred in one pup in the low dose group in the F2 generation which was dead at the initial litter examination. Visceral findings on this pup were anopthalmia, short jaw, small left atria, septum of the heart ventricles absent, and tissue adema. The other visceral findings in this study were slight variations from normal or slightly delayed development. No lesions were observed which were attributable to treatment. Lesions which were observed were generally slight to mild, and were considered to be incidental findings of little or no clinical significance. Terminal body weights of the F1 females in the low and high dose groups and F1 males in the high dose group were significantly lower than the control groups. 

Conclusions:

Nitroguanidine, at dose levels from 1.3 to 12.7 parts per thousand in the diet fed continously to parental male and female Sprague-Dawley rats starting at 56 to 58 days of age through weaning of the F2 generation, did not cause reproductive or fertility toxicologic effects under the conditions of this study. In young adult rats these dose levels approximated the 100, 316, and 1000 mg/kg/day nitroguanidine dose levels tested in developmental toxocity studies in rats and rabbits.

Executive summary:

In a 2-generation reproduction study Nitroguanidine was administered to 25 Sprague-Dawley rats/sex/dose in diet at dose levels of 0, 1300, 4000, 12700 ppm (equals 0, 100, 316, or 1000 mg/kg bw/day in young adults).

Nitroguanidine caused a decrease in some of the weekly body weights in the high dose F1 males and females and low dose F1 females. There were no dose-related effects on clinical signs, mating, fertility, gestation, litter size, pup weights or survival. Histopathological examination of the reproductive organs on adult animals and gross examination of weanlings showed no lesions attributable to nitroguanidine in any of the generations. The NOAEL is 12700 ppm ( ~1000 mg/kg bw/day). 

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study similar to OECD 416 in rat.  

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available (further information necessary)

Effect on fertility: via dermal route

Endpoint conclusion:

no study available (further information necessary)

Additional information

In a fertility and reproductive study in rats, nitroguanidine was administered at dose levels of 1.3, 4.0, and 12.7 ppt in the diet (equals 100, 316, and 1000 mg/kg/day in young adult rats). Nitroguanidine was well tolerated at all dose levels. One adult animal in the low dose group, an F1 male, died during the study. This was not considered to be substance related. Nitroguanidine had a slight effect on adult body weight. Some of the weekly body weights of high dose group animals were significantly lower than those of the controls, but the high dose group animal weights were not consistently lower throughout any of the study periods. Terminal body weights at necropsy were significantly lower than the controls for the F1 high dose males and low and high dose F1 females. Nitroguanidine also had as light effect on food consumption. Reduced food consumption occurred in the high dose group for a few sporadic weeks during the study. Nitroguanidine did not affect clinical signs. All of the F1 generation dose groups, including the controls, had a higher incidence of irritability than the parental animals. Nitroguanidine did not affect the reproductive or live pup indices. The parental generation litters had lower viability and lactation indices in the low dose group and the mid dose group had a lower lactation index than the controls. Because there were no differences in these indices for the high dose group in either generation, the decreased survival does not appear to be dose-related. Nitroguanidine did not affect the pup weights, litter weights, initial examination findings, or visceral examination findings on culled or dead pups. Histopathological examination of the reproductive organs of the adult animals in the control and high dose groups and gross examination of weanlings showed no lesions attributable to nitroguanidine in any of the generations.

Nitroguanidine, at dose levels from 1.3 t o 12.7 parts per thousand in the diet fed continuously to parental male and female Sprague-Dawley rats starting at 56 to 58 days of age through to the weaning of the F2 generation, did not cause reproductive or fertility-toxicologic effects under the conditions of this study. In young adult rats these dose levels approximated the 100, 316, and 1000 mg/kg/day.

Short description of key information:
The assessment of toxicity to reproduction is based on a 2-generation study in Sprague-Dawley rats similar to OECD 416 but without determination of sperm parameters.

Justification for selection of Effect on fertility via oral route:
Reliable guideline study

Effects on developmental toxicity

Description of key information

The assessment of developmental toxicity is based on two teratogenicity studies in Sprague-Dawley rats and

New Zealand white rabbits, respectively, comparable to OECD 414. The incidence of spontaneous malformations in rabbits and rats is similar to historical controls. Therefore, Nitroguanidine is not considered to be teratogenic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

other: LAIR OP-STX-40; Developmental Toxicity Study

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

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

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

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

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.

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.

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

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1986 to February 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: : LIAR OP-STX-40 "Developmental Toxicity Study"

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP study protocol 86003

Limit test:

no

Specific details on test material used for the study:

Test substance supplier Sunflower AAP
Assey 99.2%

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, Watsonville, CA
- Age at study initiation: males: proven breeders, approx. 5.5 months; females: nulliparous 4 months at arrival and 5 months at start of breeding
- Weight at study initiation : PHASE I: males: 3.8-4.7 kg; females: 3.1-4.3 kg; PHASE II: males: 4.2-4.7 kg, females: 3.1-4.3 kg
- Housing: individually instainless steel wire mesh cages with automatic water dispensers without bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimatisation period: 3 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14-21 °C
- Humidity (%): 42-78%
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

carboxymethylcellulose 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:

Homogeneity
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%.

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:

Each female was bred randomly to two males. Mating was confirmed by observation of the pair mating. Immediately after the first mating, the female was removed from the male’s cage and placed with another male. After the second mating the bred female was returned to her cage. Day 0 for each female was the day of mating.

Duration of treatment / exposure:

12 days

Frequency of treatment:

daily from day 6 through day 18 of gestation

Duration of test:

Phase I: 20 Oct 1986 through 15 Nov 1986
Phase II: 18 Jan 1987 through 8 Feb 1987

No. of animals per sex per dose:

100 mg/kg/day: 15
316 mg/kg/day: 15
1000 mg/kg/day: 18

Control animals:

yes, concurrent no treatment

Details on study design:

- Rationale for animal assignment (if not random): random (according to OP-STX-78)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from day 0 through day 29

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 12, 18, 23, and 29

FOOD CONSUMPTION: 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- The fetuses, uterus, and ovaries were removed, the corpora lutea were counted and the dam was examined for gross visceral signs of toxocity and reweighed. Each fetus was weighed, measured crow-to-rump, and examined externally. Fetuses were placed in 70% ethanol and then carefully eviscerated. The viscera were examined for anomalies, and the sex of the fetus was determined. The fetuses were then processed by the alizarin red S staining technique of Crary.

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 resorptions: Yes
- Other: fetus weight

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Viscera examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- External examinations: Yes: [all per litter]

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 were compared by the nonparametric Kruskal-Wallis test. If the Kruskal-Wallis test was significant, an appropriate multiple comparison test was used to determine which groups were different. The numbers of litters per group with resorptions, litters with dead fetuses, litters containing fetuses with skeletal or any variations, and the number of fetuses with skeletal or any variations were compared by chi-square analysis, and, if these were significant, the Marascuilo’s method of multiple comparison was used to determine which groups were different. The numbers of fetuses or litters with malformations or external or visceral variations were not compared statistically because there were too few.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
The predominant sign of maternal toxicity observed in this study was death in ten animals (six animals died and four moribund animals were terminated) in the 1000 mg/kg/day group. It is doubtful that these deaths were attributable to a direct pharmacological effect. Necropsy findings on the ten animals were varied with no finding common to all. The general failure to thrive of these animals suggested that the high concentrations of nitroguanidine necessary to administer the 1000 mg/kg/day dose by oral intubation interfered with the digestive processes of the animals in this group. This is supported by the decreased food consumption and weight loss during the treatment period. Additionaliy, one animal had a ruptured stomach, another animal with two masses of dosing material in the stomach had a ruptured esophagus. The tubing used to administer the test compound was smooth and flexible, not likely to tear healthy tissue. Nitroguanidine also reduced food consumption and decreased body weight in rats when given by oral gavage at 1000 mg/kg/day. Necropsy findings on several rabbits with convulsions suggested that Encephalitozoon cuniculi was a possible etiology. Stress was indicated as a contributing factor in the death of the animal with severe lymphoid depletion. Possible haemoglobinuria could have occurred as a result of the test compound in the animal with nephrosis. The only dose-related adverse maternal effects which occurred in the 100 or 316 mg/kg/day groups were a low frequency incidence of orange-rust coloured urine and thick and foamy urine.

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

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The incidence of spontaneous malformations in this study is similar to that published and to historical controls from this laboratory and is not attributed to nitroguanidine.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Developmental toxicity

The four primary manifestations of developmental toxicity are death of the conceptus, malformation, retarded development, and functional deficit. This study was designed to screen for the first three. In a developmental toxicity test the fetal examination findings may range in severity from slightly retarded development or minor variations to major malformations. Retarded development may be transitory, for example, caused by decreased maternal food consumption, and the retarded offspring may catch up quickly after birth or after weaning. Minor variations from normal may not have an adverse effect on the function and quality of life of the offspring. Major structural malformations, such as malformed or missing organs or limbs, can either be life threatening or severely limit the functioning and longevity of the offspring. A test substance is considered developmentally toxic if, when administered at a dose level that is not overtly maternally toxic, it produces malformations at a significantly higher incidence than in the controls. Although variations are not as serious as malformations, a significantly increased incidence of variations, in comparison to the controls, is a sign of some fetal or maternal toxicity. Spontaneous malformations are those that occur randomly, usually at low frequency, and are of unknown cause, and whose incidence is not dose-related. Nitroguanidine increased the number of litters with resorptions in the 100 mg/kg/day and 1000 mg/kg/day groups and the percent resorption per litter in all dose groups in comparison to the control group. One dam in the 1000 mg/kg/day group had 100% of implants resorbed. An increased incidence of resorptions is a manifestation of developmental toxicity. In this study each fetus was examined externally at cesarean section and then for visceral and skeletal abnormalities. The findings on each fetus were described and categorized as either variations or malformations, depending on the severity or whether the changes were permanent. The finding of 13 ribs (unilateral, bilateral, or rudimentary) was not included in this report because it occurred at a high frequency and was not dose-related. Those findings categorized as variations (included such transitory findings as retarded ossification includes those fetuses with fewer than six sternebrae, fewer than 15 phalanges per forepaw, and fewer than 12 phalanges per hindpaw ossified) and minor deviations from normal that may or may not be permanent such as slightly misshapen sternebrae or ribs, extra dots of ossification, dilated renal pelvis, elongated ovaries, enlarged heart ventricle, and bloated abdomen. Findings of a more serious consequence that were categorized as malformations were left palate, displaced ureter, and ectrodactyly.

The retarded development in the 1000 mg/kg/day group is resulted in fetuses that were significantly lighter in weight and had an increased incidence of skeletal variations in comparison to the controls. This retarded development could be attributed to maternal toxicity rather than to a direct effect of nitroguanidine on the fetus. The 1000 mg/kg/day group dams lost weight and consumed less food than the controls during the treatment period. The malformations observed in this study are considered spontaneous because they are not dose-related and occurred at a low frequency. One dam in the 1000 mg/kg/day group had nasal discharge from Days 7 through 29, and marked necrosis of the lung was observed at cesarean section. The fetus with multiple malformations (cleft palate and displaced ureter), the fetus with bloated abdomen at external examination, and the fetus with enlarged heart ventricle at visceral examination were in her litter. The malformations and variations which occurred in this litter could be attributed to the compromised condition of the dam. Two fetuses (one in the 100 mg/kg/day group and one in the 1000 mg/kg/day group) out of a total of 474 fetuses in the study were malformed.

The incidence of spontaneous malformations in this study is similar to published and historical controls from this laboratory and is not attributed to nitroguanidine.

Effect of Nitroguanidine on Survival and Pregnancy
	Nitroguanidine (mg/kg/day):
Examination Finding:	0	100	316	1000
Bred females	17	18	16	22
Females that died*	0	0	0	10
Nongravid	0	0	0	1
Gravid	0	0	0	7
Pregnancy not confirmed	0	0	0	2
Females examined on Day 29	17	18	16	12
Nongravid	4	3	1	1
Gravid	13	15	15	11
With live fetuses	13	15	15	10
With dead fetuses	0	3	1	2
With resorptions	3	13 #	7	5 #
Resorptions only	0	0	0	1
Females that were gravid	13	15	15	18

*Includes females that were euthanized in a moribund condition.

# Significantly different from control by Marascuilo's method of multiple comparison of proportions, p < 0.05.           

Conclusions:

There was no evidence of nitroguanidine producing teratogenicity (malformations) in rabbits under conditions of this study. Nitroguanidine causes an increase of number of litters with resorptions and percent resorption per litter in all dose groups. This effect occurs at dose level that does not produce overt maternal toxicity but can be attributed to a low resorption rate in the concurrent control. Compared to historical data there is no significantly deviation at 100 and 316 mg/kg bw/day.

Executive summary:

In a developmental toxicity study Nitroguanidin (100%) was administered to 15-18 female New Zealand White rabbits/dose by gavage at dose levels of 0, 100, 316, or 1000 mg/kg bw/day from days 6 through 18 of gestation.

The maternal LOAEL is 1000 mg/kg bw/day, based on weight loss and decreased food consumption. The maternal NOAEL is 316 mg/kg bw/day. 

An increased number of litters with resorptions were reported in the 100 mg/kg/day and 1000 mg/kg/day groups and the percent resorption per litter were increased in all dose groups. One dam in the 1000 mg/kg/day group had 100% of implants resorbed. However, the control group in this study had a lower resorption rate than published reports for NZW rabbits and the resorption rate found in the nitroguanidine dosed animals was within these published control values. Due to the historical data on resorption rates the findings in the developmental study in rabbits are not considered to be strong enough. The developmental toxicity NOAEL for nitroguanidine in rabbits is 316 mg/kg/day based on maternal toxicity.

The incidence of spontaneous malformations in this study is similar to published and historical controls from this laboratory. Nitroguanidine is not considered to be teratogenic. 

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available (further information necessary)

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available (further information necessary)

Additional information

1) In a developmental toxicity study in rats, nitroguanidine given by oral gavage on gestational days 6 through 15 at 1000 mg/kg/day produced decreased food consumption, maternal weight loss, and smaller foetuses with an increased incidence of retarded ossification of the sternebrae, caudal vertebrae, and pubis. No teratogenic effects have been reported. The developmental toxicity NOAEL for nitroguanidine in rats is 1000 mg/kg/day. Foetotoxicity at 1000 mg/kg/d can be attributed to maternal toxicity. The NOAEL of foetotoxicity is 316 mg/kg/d based on maternal toxicity.

2) In a developmental toxicity study in NZW rabbits, nitroguanidine given by oral gavage on gestation days 6 through 18 at 1000 mg/kg/day caused death or termination in a moribund condition in 10 of 22 animals. The animals at this dose level exhibited weight loss and decreased food consumption. Fetuses from these dams were lighter in weight and had an increased incidence of retarded ossification of the sternebrae, olecranon, patellae, and phalanges. The fetotoxicity NOAEL for nitroguanidine in rats is 316 mg/kg/day based on maternal toxicity. This result is in accordance with the findings in rat as second species.

In addition an increased incidence of resorptions occurred in rabbits in all dose groups (100, 316, and 1000 mg/kg/day) versus the control group. However, the control group in this study had a lower resorption rate than published reports for NZW rabbits and the resorption rate found in the nitroguanidine dosed animals was within these published control values. The authors of the study concluded that nitroguanidine did not have teratogenic potential, but had equivocal evidence of fetotoxicity in rabbits. Due to the historical data on resorption rates the findings in the developmental study in rabbits are not considered to be strong enough to challenge the results in rats. The teratogenic toxicity NOAEL for nitroguanidine in rabbits is 1000 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via oral route:
GLP, comparable to OECD 414 in rat and rabbit

Justification for classification or non-classification

Nitroguanidine, at dose levels up to 1000 mg/kg bw/day in diet did not cause toxic effects to reproduction or fertility under the conditions of a 2-generation study in SD rats.

In two developmental toxicity studies in rats and rabbits, respectively, nitroguanidine administered by oral gavage on gestational days 6 through 15 caused maternal toxicity at the highest dose level. This results contradicts the 2-generation study but can be attributed to the difference between administration by diet and bolus administration by gavage. 

No teratogenic effects have been reported. Foetotoxicity of nitroguanidine is caused only at doses of overt maternal toxicity and can be attributed to these effects. Thus nitroguanidine is not classified as a developmental toxicant.