N-butylphosphorothioic triamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Additional information

In a preliminary study of the effect on pregnancy of the rat, it was concluded that 500 mg/kg/day is the highest dosage that can be tolerated by the pregnant rat. Accordingly, this dosage would be a suitable high dosage level for future investigation of embryofoetal toxicity.

Introduction: The objective of this study was to assess the effects of continuous dietary administration of N-(n-butyl phosphorothioic triamide) (NBPT), an organophosphorrus compound, upon the reproductive performance of the male and female rats of the CD strain through two successive generations. The study was conducted in accordance with the regulations or guidelines of the following authorities:

- Draft guidelines of the Environmental Protection Agency of United States of America OPPTS 870.3800 (1984) (N.B: Revisions of the draft guidelines released as a "Public Draft" (February 1996) were taken into account).

- Organisation for Economic Co-operation and Development, Guideline 416 (1983)

- Ministry of Agriculture, Forestry and Fisheries of Japan (1985)

Method:

The NBPT was administered continuously in the diet at concentrations of 200, 800 or 3200 ppm to groups of rats to the two generations. A fourth group received the basal diet without the test material and served as the Control.

The F0 generation, which comprised 32 males and 32 females in each group, received the treated diet for 10 weeks before pairing and throughout mating, gestation and lactation. From the Fl litters, 32 male and 32 female offspring were selected to fonn the F1 generation. Both sexes received treatment for a minimum of 10 weeks from selection, throughout pairing, gestation and lactation. Pup survival, body weight, development, and sexual maturation were evaluated.

All F0 and F1 adult animals were subjected to a detailed necropsy and the reproductive organs and the brain, liver, kidneys, adrenals and spleen were weighed and retained. Sperm counts, motility and morphology were determined for the left epididymis and testis for all F0 and F1 males. Histopathological examinations were performed on reproductive organs from Control and high dosage animals. In addition, the retained epididymis was examined for all F0 and F1 males in the 800 ppm and 200 ppm groups.

Non-selected F1 offspring were subjected to a necropsy examination on Day 35 of age and the brain, spleen, thymus, ovaries and testes weighed. All non-selected F2 offspring were subjected to a necropsy examination on Day 35 of age and the brain, spleen and thymus were weighed.

Principal findings

Non-reproductive phases

The inclusion of NBPT in the diet at levels up to 3200 ppm did not affect the general condition of the animals and there were no mortalities considered to be related to treatment.

Treatment of F0 males at 3200 ppm and, of F0 females at 3200 and 800 ppm, was associated with lower weight gains prior to pairing. Bodyweight gain of F1 and F2 offspring at 3200 ppm and of F1 offspring at 800 ppm was retarded from Day 14 of age compared with Controls. The overall growth rate of selected F1 animals from selection to termination was also retarded at 3200 ppm.

F0 and F1 females receiving 3200 ppm showed lower weight gains during gestation and did not show the characteristic pattern of late lactation weight loss.

There was no effect of treatment on pup development or sexual maturation of F1 animals.

Achieved intake of NBPT

In the F0 generation at 3200 ppm, chemical intake was approximately 370 mg/kg/day for males and females during the first week of treatment. In the week before pairing, intake had declined naturally to approximately 180 mg/kglday for males and 220 mg/kg/day for females. Chemical intake for females was higher during gestation and increased to a maximum of around 530 mg/kglday during Days 7-13 of lactation at peak physiological demand.

At 3200 ppm during the first week of the F1 generation, chemical intake was approximately 480 mg/kg/day for males and females. In the week before pairing, intake was about 200 mg/kg/day for males and 250 mg/kg/day for females. Chemical intake for females was higher during early gestation and increased to around 520 mg/kg/day during Days 7-13 of lactation at peak physiological demand.

Chemical intake by animals in the lower treatment groups was in proportion to the dietary concentrations but followed the same patterns as at 3200 ppm.

Fertility and reproductive performance

In both the F0 and F1 generations, there were no adverse effects of treatment on mating performance.

At 3200 ppm only, mean implantation count and total and live litter size on Day 1 of age was slightly lower than in Controls on both generations.

There was no effect of treatment on the parturition process or, peri and post-natal survival of the offspring. Pre-weaning developmental landmarks were unaffected and despite a retardation in growth rate among F1 offspring at 3200 ppm, the attainment of sexual maturity was not delayed.

Terminal studies

Treatment of F0 males at 3200 ppm was associated with a significantly lower percentage of progressively motile sperm in the left epididymis compared with Controls and, further analysis demonstrated reductions in straight line and curvilinear velocity of the sperm. Organ weight analysis showed a higher bodyweight relative weight for the epididymides at 3200 ppm. Microscopic examination of the right epididymis detected a specific treatment-related change for all males at 3200 ppm and some males at 800 ppm: cells lining the epididymal duct in the distal part of the caput and the proximal portion of the corpus epididymis exhibited macro and microvesiculation which resembled fat vacuoles.

Treatment of F1 males at 3200 ppm was associated with significantly lower percentages of motile and progressively motile sperm in the left epididymis compared with Controls. At 800 ppm, the percentage of motile sperm was lower than in Controls Further analysis demonstrated a significant reduction in the percentage of 'rapid' sperm at 3200 ppm only. As in the Fa generation, organ weight analysis showed a higher bodyweight relative weight for the epididymides at 3200 ppm and microscopic examination of the right epididymis detected the same vacuolation of a defined area of the epithelium at 3200 and 800 ppm as had been seen in F0 males. 200 ppm was a no-effect-Ievel for the epididymis.

There was a clear increase in the incidence of F1 females in the 3200 ppm group exhibiting atrophic and mucified vaginal epithelium.

Conclusion

Adult toxicity:

The No-observed-effect-Ievel (NOEL) for toxicity to the F0 and F1 adult animals was 200 ppm (11 and 14 mg/kg/day for F₀males and females respectively based on Week 10 bodyweights or, 12 and 16 mg/kg/day for F1 males and females respectively) based on decreased bodyweight gain at 3200 ppm in males and females and at 800 ppm in females; decreased sperm motility at 3200 ppm in the F0 males and at 3200 and 800 ppm in the F1 males, and epididymal lesions at 3200 and 800 ppm in males. There were no treatment-related effects on mortality, clinical signs of toxicity, food consumption or food efficiency.

Reproductive toxicity:

The NOEL for reproductive toxicity was 800 ppm (approximately 43-61 mg/kg/day based on Week 10 bodyweights) based on slightly dccreased irnplantation count and litter size for the F0 and F1 females at 3200 ppm, and delayed or non-recovery of oestrus cyclicity postpregnancy of F1 females at 3200 ppm. Neither mating or fertility indices were affected at any dose. There were no adverse effects on testicular or ovarian function at 3200 ppm.

Pup toxicity:

The NOEL for toxicity to pups was 200 ppm (approximately 32 mg/kg/day achieved maternal dosage during Days 7-13 of lactation) based on bodyweight gain reductions occurring after the onset of self-feeding of treated diets in F1 pups at 3200 and 800 ppm and in F2 pups at 3200 ppm. There were no treatment-related effects on pup survival, development, or sexual maturation.

Short description of key information:
The objective of this study was to assess the effects of continuous dietary administration of N-(n-butyl phosphorothioic triamide) (NBPT), an organophosphorrus compound, upon the reproductive performance of the male and female rats of the CD strain through two successive generations.

Adult toxicity:
The No-observed-effect-Ievel (NOEL) for toxicity to the F0 and F1 adult animals was 200 ppm (11 and 14 mg/kg/day for F0males and females respectively based on Week 10 bodyweights or, 12 and 16 mg/kg/day for F1 males and females respectively) based on decreased bodyweight gain at 3200 ppm in males and females and at 800 ppm in females; decreased sperm motility at 3200 ppm in the F0 males and at 3200 and 800 ppm in the F1 males, and epididymal lesions at 3200 and 800 ppm in males. There were no treatment-related effects on mortality, clinical signs of toxicity, food consumption or food efficiency.

Reproductive toxicity:
The NOEL for reproductive toxicity was 800 ppm (approximately 43-61 mg/kg/day based on Week 10 bodyweights) based on slightly dccreased irnplantation count and litter size for the F0and F1females at 3200 ppm, and delayed or non-recovery of oestrus cyclicity postpregnancy of F1females at 3200 ppm. Neither mating or fertility indices were affected at any dose. There were no adverse effects on testicular or ovarian function at 3200 ppm.

Pup toxicity:
The NOEL for toxicity to pups was 200 ppm (approximately 32 mg/kg/day achieved maternal dosage during Days 7-13 of lactation) based on bodyweight gain decreases occurring after the onset of self-feeding of treated diets in F1 pups at 3200 and 800 ppm and in F2 pups at 3200 ppm. There were no treatment-related effects on pup survival, development, or sexual maturation.

Effects on developmental toxicity

Description of key information

Two studies were performed, one in the rat and the other in the rabbit, to assess developmental toxicity/teratogenicity:
1) N-(n-butyl) thiophosphoric triamide (NBPT) administered by oral gavage to time-mated female rat from Day 6 through to Day 15 of pregnancy inclusive.
500 mg/kg/day is a no adverse effect level forin uterodevelopment of the conceptus and, there was no evidence for any selective toxicity to the conceptus. 
2) N-(n-butyl) thiophosphoric triamide (NBPT) administered by oral gavage to time-mated female rabbits from Day 6 through to Day 18 of pregnancy inclusive.
200 mg/kg/day, is a no adverse effect level for in uterodevelopment of the conceptus and, there was no evidence for any selective toxicity to the conceptus.

Additional information

In a preliminary study of the effect on pregnancy of the rat, it was concluded that 500 mg/kg/day is the highest dosage that can be tolerated by the pregnant rat. Accordingly, this dosage would be a suitable high dosage level for future investigation of embryofoetal toxicity.

DEVELOPMENTAL TOXICITY/TERATOGENICITY

Two studies were performed, one in the rat and the other in the rabbit, to assess developmental toxicity/teratogenicity:

1) The study was performed to assess the effect of N-(n-butyl) thiophosphoric triamide (NBPT), a urease inhibitor, upon the pregnant rat and her unborn offspring, when administered by oral gavage to time-mated females from Day 6 through to Day 15 of pregnancy inclusive.

Within the context of this study, it was concluded that 500 mg/kg/day is a no adverse effect level for in utero development of the conceptus and, there was no evidence for any selective toxicity to the conceptus. At 125 mg/kg/day, other than 1/25 animals showing noisy respiration and 8/25 animals showing post-dose salivation, there was no obvious maternal response to treatment.

2)The study was performed to assess the effect of N-(n-butyl) thiophosphoric triamide (NBPT), a urease inhibitor, upon the pregnant rabbit and her unborn offspring, when administered by oral gavage to time-mated females from Day 6 through to Day 18 of pregnancy inclusive.

Within the context of this study, it was concluded that 200 mg/kg/day, is a no adverse effect level for in utero development of the conceptus and, there was no evidence for any selective toxicity to the conceptus. The no adverse effect level for the parent female is 50 mg/kg/day.

Justification for classification or non-classification

TOXICITY TO REPRODUCTION

In a preliminary study of the effect on pregnancy of the rat, it was concluded that 500 mg/kg/day is the highest dosage that can be tolerated by the pregnant rat. Accordingly, this dosage would be a suitable high dosage level for future investigation of embryofoetal toxicity.

Introduction: The objective of this study was to assess the effects of continuous dietary administration of N-(n-butyl phosphorothioic triamide) (NBPT), an organophosphorrus compound, upon the reproductive performance of the male and female rats of the CD strain through two successive generations. The study was conducted in accordance with the regulations or guidelines of the following authorities:

- Draft guidelines of the Environmental Protection Agency of United States of America OPPTS 870.3800 (1984) (N.B: Revisions of the draft guidelines released as a "Public Draft" (February 1996) were taken into account).

- Organisation for Economic Co-operation and Development, Guideline 416 (1983)

- Ministry of Agriculture, Forestry and Fisheries of Japan (1985)

Method:

The NBPT was administered continuously in the diet at concentrations of 200, 800 or 3200 ppm to groups of rats to the two generations. A fourth group received the basal diet without the test material and served as the Control.

The F0 generation, which comprised 32 males and 32 females in each group, received the treated diet for 10 weeks before pairing and throughout mating, gestation and lactation. From the Fl litters, 32 male and 32 female offspring were selected to fonn the F1 generation. Both sexes received treatment for a minimum of 10 weeks from selection, throughout pairing, gestation and lactation. Pup survival, body weight, development, and sexual maturation were evaluated.

All F0 and F1 adult animals were subjected to a detailed necropsy and the reproductive organs and the brain, liver, kidneys, adrenals and spleen were weighed and retained. Sperm counts, motility and morphology were determined for the left epididymis and testis for all F0 and F1 males. Histopathological examinations were performed on reproductive organs from Control and high dosage animals. In addition, the retained epididymis was examined for all F0 and F1 males in the 800 ppm and 200 ppm groups.

Non-selected F1 offspring were subjected to a necropsy examination on Day 35 of age and the brain, spleen, thymus, ovaries and testes weighed. All non-selected F2 offspring were subjected to a necropsy examination on Day 35 of age and the brain, spleen and thymus weighed.

Principal findings

Non-reproductive phases

The inclusion of NBPT in the diet at levels up to 3200 ppm did not affect the general condition of the animals and there were no mortalities considered to be related to treatment.

Treatment of F0 males at 3200 ppm and, of F0 females at 3200 and 800 ppm, was associated with lower weight gains prior to pairing. Bodyweight gain of F1 and F2 offspring at 3200 ppm and of F1 offspring at 800 ppm was reduced from Day 14 of age compared with Controls. The overall growth rate of selected F1 animals from selection to termination was also reduced at 3200 ppm.

F0 and F1 females receiving 3200 ppm showed lower weight gains during gestation and did not show the characteristic pattern of late lactation weight loss.

There was no effect of treatment on pup development or sexual maturation of F1 animals.

Achieved intake of NBPT

In the F0 generation at 3200 ppm, chemical intake was approximately 370 mg/kg/day for males and females during the first week of treatment. In the week before pairing, intake had declined naturally to approximately 180 mg/kglday for males and 220 mg/kg/day for females. Chemical intake for females was higher during gestation and increased to a maximum of around 530 mg/kglday during Days 7-13 of lactation at peak physiological demand.

At 3200 ppm during the first week of the F1 generation, chemical intake was approximately 480 mg/kg/day for males and females. In the week before pairing, intake was about 200 mg/kg/day for males and 250 mg/kg/day for females. Chemical intake for females was higher during early gestation and increased to around 520 mg/kg/day during Days 7-13 of lactation at peak physiological demand.

Chemical intake by animals in the lower treatment groups was in proportion to the dietary concentrations but followed the same patterns as at 3200 ppm.

Fertility and reproductive performance

In both the F0 and F1 generations, there were no adverse effects of treatment on mating performance.

At 3200 ppm only, mean implantation count and total and live litter size on Day 1 of age was slightly lower than in Controls on both generations.

There was no effect of treatment on the parturition process or, peri and post-natal survival of the offspring. Pre-weaning developmental landmarks were unaffected and despite a retardation in growth rate among F1 offspring at 3200 ppm, the attainment of sexual maturity was not delayed.

Terminal studies

Treatment of F0 males at 3200 ppm was associated with a significantly lower percentage of progressively motile sperm in the left epididymis compared with Controls and, further analysis demonstrated reductions in straight line and curvilinear velocity of the sperm. Organ weight analysis showed a higher bodyweight relative weight for the epididymides at 3200 ppm. Microscopic examination of the right epididymis detected a specific treatment-related change for all males at 3200 ppm and some males at 800 ppm: cells lining the epididymal duct in the distal part of the caput and the proximal portion of the corpus epididymis exhibited macro and microvesiculation which resembled fat vacuoles.

Treatment of F1 males at 3200 ppm was associated with significantly lower percentages of motile and progressively motile sperm in the left epididymis compared with Controls. At 800 ppm, the percentage of motile sperm was lower than in Controls Further analysis demonstrated a significant reduction in the percentage of 'rapid' sperm at 3200 ppm only. In the F1 generation, organ weight analysis showed a higher bodyweight relative weight for the epididymides at 3200 ppm and microscopic examination of the right epididymis detected the same vacuolation of a defined area of the epithelium at 3200 and 800 ppm as had been seen in F0 males. 200 ppm was a no-effect-Ievel for the epididymis.

There was a clear increase in the incidence of F1 females in the 3200 ppm group exhibiting atrophic and mucified vaginal epithelium.

Conclusion

Adult toxicity:

The No-observed-effect-Ievel (NOEL) for toxicity to the F0 and F1 adult animals was 200 ppm (11 and 14 mg/kg/day for F₀males and females respectively based on Week 10 bodyweights or, 12 and 16 mg/kg/day for F1 males and females respectively) based on decreased bodyweight gain at 3200 ppm in males and females and at 800 ppm in females; decreased sperm motility at 3200 ppm in the F0 males and at 3200 and 800 ppm in the F1 males, and epididymal lesions at 3200 and 800 ppm in males. There were no treatment-related effects on mortality, clinical signs of toxicity, food consumption or food efficiency.

Reproductive toxicity:

The NOEL for reproductive toxicity was 800 ppm (approximately 43-61 mg/kg/day based on Week 10 bodyweights) based on slightly dccreased irnplantation count and litter size for the F₀and F₁females at 3200 ppm, and delayed or non-recovery of oestrus cyclicity postpregnancy of F₁females at 3200 ppm. Neither mating or fertility indices were affected at any dose. There were no adverse effects on testicular or ovarian function at 3200 ppm.

Pup toxicity:

The NOEL for toxicity to pups was 200 ppm (approximately 32 mg/kg/day achieved maternal dosage during Days 7-13 of lactation) based on bodyweight gain decreases occurring after the onset of self-feeding of treated diets in F1 pups at 3200 and 800 ppm and in F2 pups at 3200 ppm. There were no treatment-related effects on pup survival, development, or sexual maturation.

DEVELOPMENTAL TOXICITY/TERATOGENICITY

Two studies were performed, one in the rat and the other in the rabbit, to assess developmental toxicity/teratogenicity.

1) The study was performed to assess the effect of N-(n-butyl) thiophosphoric triamide (NBPT), a urease inhibitor, upon the pregnant rat and her unborn offspring, when administered by oral gavage to time-mated females from Day 6 through to Day 15 of pregnancy inclusive.

Within the context of this study, it was concluded that 500 mg/kg/day is a no adverse effect level forin uterodevelopment of the conceptus and, there was no evidence for any selective toxicity to the conceptus. At 125 mg/kg/day, other than 1/25 animals showing noisy respiration and 8/25 animals showing post-dose salivation, there was no obvious maternal response to treatment.

2) The study was performed to assess the effect of N-(n-butyl) thiophosphoric triamide (NBPT), a urease inhibitor, upon the pregnant rabbit and her unborn offspring, when administered by oral gavage to time-mated females from Day 6 through to Day 18 of pregnancy inclusive.

Within the context of this study, it was concluded that 200 mg/kg/day, is a no adverse effect level for in utero development of the conceptus and, there was no evidence for any selective toxicity to the conceptus. The no adverse effect level for the parent female is 50 mg/kg/day.