1-nitroguanidine

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

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)

Test guideline 2

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

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
Assey 99.7%

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

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

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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

Effect levels (F1)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Any other information on results incl. tables

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. 

Applicant's summary and conclusion

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.