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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 2 November 1994 and 2 August 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standardtest guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20 September 1994 to 5 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
GLP compliance:
yes
Limit test:
no
Justification for study design:
Dosages were based on findings in a 15-day repeat dose rat study where effects at 1000 mg/kg/day included occasional signs of increased salivation and languinal behaviour, decrease in blood urea, nitrogen and alanine aminotransferase. Reductions in brain and erythrocyte cholinesterase were also observed.

Oral administration was chosen since the oral route is a likely route of human exposure.

The rat was used since this is a universally accepted species in reproductive studies and additionally, the testing facility has background data on reproductive performance in embryofoetal toxicity studies for the strain used.
Specific details on test material used for the study:
Identity: NBPT
Chemical name: N-(n-butyl) thiophosphoric triamide
Appearance: Light tan waxy solid
Storage conditions: Ca 4 deg.C in the dark
Stability of formulations: 24 hours
Species:
rat
Strain:
other: Crl: CD®BR VAF/Plus strain
Sex:
female
Details on test animals and environmental conditions:
Parent animals:
A total of 34 sexually mature (8 - 10 weeks old, weight range on arrival 199 - 245 g) Specific Pathogen Free female rats which were time-mated to identified males of the same strain were ordered from Charles River UK Limited. The day of mating, as judged by the appearance of sperm in the vaginal smear or by the presence of a vaginal plug, was considered as Day 0 of pregnancy.

Animals were weighed on arrival (Day 2 of pregnancy), examined for abnormalities and signs of ill health and thirty rats (weight range 199 - 235 g) assigned to five groups by computerised stratified randomisation to give approximately equal initial group mean bodyweights. Following allocation, the animals were earmarked to give individual identification. Prior to the commencement of treatment, the health status of the animals was reviewed by a Veterinary Officer and considered to be satisfactory for the study.

Animal room controls for temperature and humidity were set at 21 deg.C and 55% respectively. Recorded values were within the ranges 21 ± 5 deg.C and 59 ± 14% respectively. Lighting was controlled to give 12 hours light (0800 - 2000 hours) and 12 hours dark per 24 hours.

Throughout the study the animals were housed three to a cage in suspended stainless steel cages equipped with solid sides and wire grid front, back, front and top.

The cages constituting each treatment group were dispersed so that possible environmental influences from their spatial distribution would be, as far as possible, equilibrated.

Throughout the study, each cage was identified by a label coloured according to the group and recording the study schedule number, animal numbers, details of treatment and the names of the Study Supervisor and Director.

All animals were given free access to Special Diet Services (SOS) Laboratory Animal Diet No. 1 and to tap water via water bottles.
Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol 300 in water
Details on exposure:
The test material was weighed out and dissolved in PEG 300, the volume of PEG used being half that of the required final volume. Dissolution was achieved by using a mortar and pestle followed by a homogeniser. This solution was then made up to volume using distilled, deionised water giving a hazy formulation (suspension) and mixed by stirring.

Lower concentrations were made by serial dilution.

Formulations were prepared daily.

Formulations were stirred immediately prior to, and during dosing using a magnetic stirrer.
Details on mating procedure:
Animals were received time-mated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the commencement of the study, the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the homogeneity and stability of the formulations were satisfactory under the conditions of the study.

Samples were taken for analysis of achieved concentrations of the test substance from formulations prepared on the first day of treatment. As a contingency further samples were taken on Day 15; these samples were not analysed.
Duration of treatment / exposure:
10 Days
Frequency of treatment:
Daily
Details on study schedule:
Treatment by gavage commenced on Day 6 of pregnancy and continued dosing up to and including Day 15 of pregnancy.

Dosage volumes were calculated for individual animals on Day 6 of pregnancy and adjusted according to bodyweight on Day 8, 10, 12 and 14.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
6 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were weighed on arrival (Day 2 of pregnancy) and thirty rats assigned to five groups by computerised stratified randomisation to give approximately equal initial group mean bodyweights. (Adjustments were made to the group allocation in order to ensure an acceptable distribution of the males to which females were mated).
Positive control:
No
Parental animals: Observations and examinations:
All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment.

Food consumption was measured from weighday to weighday commencing on Day 3 of pregnancy.

Gravimetric daily measurement of water consumption was initiated from Day 8, after visual inspection of water bottles indicated increased consumption in the 1000 mg/kg/day group.

All animals were weighed initially (=Day 2 of pregnancy) and on Days 3, 6, 8, 10, 12, 14, 16, 18 and 20.
Litter observations:
Live young were examined externally sexed, weighed and then discarded.
Postmortem examinations (parental animals):
On Day 20 of pregnancy, the animals were killed by C02 asphyxiation, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs.
Postmortem examinations (offspring):
The ovaries and uteri were examined immediately to determine:
- Number of corpora lutea
- Number and distribution of live young
- Number and distribution of embryofoetal deaths
- Individual foetal weight from which the litter weight was calculated
- Foetal abnormalities

Embryofoetal deaths were classified as:
Early: only placenta visible at termination.
Late: both placental and embryonic remnants visible at termination.

Uteri or individual uterine horns without visible implantations were examined for evidence of implantation using a modified Salewski technique (Salewski 1964) .
Statistics:
Significance tests were not performed in view of the low group size.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 1000 mg/kg bw/day was associated with:
Several clinical signs including abnormalities of gait (unsteady, high stepping and splayed gait), reduced bodytone, wet faeces, post-dose salivation and wet coats, partially closed eyes, piloerection and wet urogenital region.


Treatment at 125, 250 and 500 mg/kg bw/day was associated with:
Post-dose salivation was observed in all treated groups; group incidences were 5 of 6, 5 of 6 and 6 of 6 animals at 125, 250 and 500 mg/kg/day respectively. This sign was first apparent on Day 7 at 500 mg/kg/day, on Day 8 at 250 mg/kg/day and on Day 10 at 125 mg/kg/day. At 500 mg/kg/day only, wet coats after dosing were observed during Days 8 to 10 and 12 to 15.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
At 125, 250 and 500 mg/kg bw/day there were no deaths.

At 1000 mg/kg bw/day 5 animals which showed loss of bodyweight were sacrificed for humane reasons due to poor condition during Days 12 to 15 of pregnancy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day lower bodyweight gains during Days 6 to 10 compared with controls. From Day 10, loss of bodyweight was recorded in 5 of 6 animals - this is reflected in a group mean loss of bodyweight during Days 10 to 12.

The 5 animals which showed loss of bodyweight were sacrificed for humane reasons due to poor condition during Days 12 to 15 of pregnancy.

Treatment at 500 mg/kg/day was associated with lower bodyweight gain during the first 4 days of treatment (Days 6 to 10) compared with the controls. Weight gains during Days 10 to 12 were slightly superior to controls. During Days 12 to 16, a second period of lower weight gains was recorded. Gains during Days 16 to 20 were comparable to controls. Overall weight gains during Days 6 to 20 were 12 % lower than in controls.

Treatment at 250 mg/kg/day was associated with a slight reduction in bodyweight gain during Days 8 to 10 compared with the controls. As at 500 mg/kg/day, although weight gains during Days 10 to 12 were comparable to controls, gains during Days 12 to 16 were lower than in controls. Gains during Days 16 to 20 were superior to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was an obvious reduction in food consumption.

At 125, 250 and 500 mg/kg bw/day there was no clear or consistent effect on food intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there is an obvious increase in water intake.

At 500 or 250 mg/kg/day water consumption was slightly increased during Days 8 to 19 compared with controls.

At 125 mg/kg/day, water intake was marginally higher than in controls throughout Days 8 to 19.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
A total of 6, 6, 6 and 5 females had live young at Day 20 of pregnancy in Groups 1 to 4 respectively and have been included in the following assessment of litter parameters.
At 1000 mg/kg bw/day, the five animals that were sacrificed for humane reasons were confirmed to be pregnant. There were no in utero deaths or macroscopic external foetal abnormalities.

There were no instances of total litter loss in utero (total resorption) at any dose.

There were no obvious adverse effects of treatment on any of the litter parameters recorded.

At 125, 250 and 500 mg/kg bw/day macroscopic external foetal examination did not indicate any adverse effect of treatment at any dosage. The only suspected malformations observed were 5 instances of suspected squat foetus syndrome in Litter No. 13 at 250 mg/kg/day. This syndrome has been observed quite frequently in rats of this strain, occurring at random within the control population. Characteristically, the syndrome tends to occur in litter based clusters and, although the aetiology of the syndrome is unknown, it is considered to be spontaneous and unrelated to treatment of the parent female.
Conclusions:
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC: Council recommendation (83/571/EEC) Annex 1 - Repeated Dose Toxicity Official Journal of the European Communities L332, 20 - 22 October 1983.
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF: Requirements for Safety Evaluation of Agricultural Chemicals published in NohSan No. 4200, Ministry of Agriculture, Forestry and Fisheries 28 January 1985.
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-(n-butyl) thiophosphoric triamide (NBPT)
- Substance type: Organophosphorous compound, a urease inhibitor
- Physical state: Light tan waxy solid
- Analytical purity: 85.8%
- Lot/batch No.: 6193-34
-Date Received: 4 August 1994
- Expiration date of the lot/batch: December 1995
-Stability of formulations: 24 hours
- Storage condition of test material: 4°C in the dark

Test animals

Species:
rat
Strain:
other: Crl: CD® BR VAF/Plus strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River UK Limited, Manston Road, Margate, Kent.

- Age at study initiation:
8 - 10 weeks old

- Weight at study initiation:
Batch A animals : weight range 213 -254 g
Batch B animals : weight range 186 - 222 g

- Fasting period before study:
No

- Housing:
The animals were housed five to a cage in suspended stainless steel cages (BiotechQl) equipped with solid sides and wire grid front, back, floor and top. The cages constituting each treatment group were dispersed within the batteries so that possible environmental influences arising from their spatial distribution were equilibrated as far as possible for all treatments.

- Diet (e.g. ad libitum):
All animals were given free access to Special Diet Services (SDS) Laboratory Animal Diet No.1

- Water (e.g. ad libitum):
All anim~l1s were given free access to tap water

- Acclimatisation period:
Not specified in the report

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
20 ± 2 °C

- Humidity (%):
65 ± 6 %

- Air changes (per hr):
Not specified in report

- Photoperiod (hrs dark / hrs light):
lighting was controlled to give 12 hours light (0800 to 2000 hours) and 12 hours dark per 24 hours

IN-LIFE DATES: From: 7 November 1994 To: 22 November 1994

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
other: PEG 300
Details on exposure:
METHOD AND FREQUENCY OF DOSE PREPARATION, FORMULATION SAMPLING AND ANALYSIS:

The test material was weighed out and dissolved in PEG 300, the volume of PEG used being half that of the required final volume. Dissolution was achieved by using a mortar and pestle followed by a homogeniser. This formulation was then placed in an ultrasonic bath, to rapidly clear air trapped in the
formulation, when this was considered necessary. This formulation was then made up to volume using distilled~ deionised water giving a hazy formulation (suspension) and mixed by stirring.

Lower concentrations were made by serial dilution.

Formulations were prepared daily.

Formulations were stirred immediately prior to, and during dosing using a magnetic stirrer.

Prior to the commencement of the study, the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the homogeneity and stability of the formulations were satisfactory under the conditions of the study. The method of analysis was supplied by the Sponsor.

Samples 'were taken for analysis of achieved concentrations of the test substance from formulations prepared on the first day of treatment. As a contingency further samples were taken towards the end of the treatment period; these samples were not analysed. Analyses were performed by the Department of Analytical Chemistry at HRC.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL PROCEDURE

Apparatus and instrumentation

High perfonnance liquid chromatograph (HPLC): As detailed in the chromatographic section.

Balances: Sartorius 1712 MP8, fitted with a data print 7381. Sartorius R2ooD, fitted with a data print 7382 2C. Mettler AT 261, fitted with a LC-45 printer.

Autodiluter: Hamilton Microlab™ 1000

Ultrasonic bath: Decon FS 300 frequency sweep.

Density meter: Stanton Redcroft PAAR DMA46

General laboratory glassware.

Typical chromatograpbic conditions

High performance liquid chromatograph (HPLC):
Pump: Perkin Elmer LC 250.
Autosampler: Perkin Elmer ISS 200.
Detector: Spectra Physics UV 1000.
Integrator: Perkin Elmer Nelson 1020.

Analytical column: Hichrom Ltd, Hypersil ODS, 5 µm, 250 x 4.6 mm id.
Guard column: Hichrom Ltd, Hypersil ODS, 5 µm, 10 x 4.6 mm id.
Column temperature: Ambient, nominally 21°C ± 1°C.
Mobile phase: Methanol/water (50/50 v/v).
Flow rate: 1.0 mI/minute.
Detector wavelength: UV, 214 nm.
Injection volume: 10 µl
Integration sensitivity: 45mV.
Retention volume: 5 mI.

Calibration
A primary standard solution was prepared for each analytical occasion by dissolving an accurately weighed quantity (25 mg) of N-(n-butyl) thiophosphoric triamide analytical standard in mobile phase. Solutions for instrument calibration, containing N-(n-butyl) thiophosphoric triamide in the concentration range 5 - 25 µg/mI, were prepared by appropriate dilution of the primary standard using mobile phase.

Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence, using the conditions detailed in the previous section.

The analytical results confirm that the doses analysed during the study were accurately formulated, that formulations were homogeneous and stable during storage (ambient temperature for 4 hours, +4°C overnight) over a 24 hour period, representing the maximum time from preparation to completion of
dosing and that the purity of the technincal grade test substance was 85.8%for the duration of the study.
Details on mating procedure:
A total of 108 sexually mature (8 - 10 weeks old, weight range on arrival 182 - 234 g) Specific Pathogen Free female rats (Crl: CD® BR VAF/Plus strain) which were time-mated to identified males of the same strain, were obtained from Charles River UK Limited, Manston Road, Margate, Kent. The first batch (A) consisted of 66 animals followed by a second batch (B) of 42 animals mated one day later. The day of mating, as judged by the appearance of spenn in the vaginal smear or by the presence of a vaginal plug, was considered as Day 0 of pregnancy..
Duration of treatment / exposure:
Treatment by gavage commenced on Day 6 of pregnancy and continued daily up to and including Day 15 of pregnancy.
Frequency of treatment:
Daily
Duration of test:
From Day 6 through to Day 15 of pregnancy.
No. of animals per sex per dose:
25 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosages employed 0 (Control), 30, 125 and 500 mg/kg/day were selected in collaboration with the Sponsor's representative (Technology ScienceGroup Inc.) based on results of a preliminary study in the pregnant rat performed at HRC (HRC report no. IMF 1/943225). In this study, treatment at
500 mg/kg/day was associated with lower bodyweight gain during pregnancy and an increase in water intake, and therefore this dosage was selected as the highest dosage level for the present study. 30 mg/kg/day was chosen as the lowest dosage for the present study as it was anticipated to be a no-effect level

- Rationale for animal assignment (if not random):
Allocation to treatment groups occurred on Day 2 of pregnancy when animals were weighed and sixty Batch A animals in the weight range 213 -254 g; and forty Batch B animals in the weight range 186 - 222 g were assigned to four :groups by computerised stratified randomisation to give approximately equal initial group mean bodyweights within each batch. (Adjustments were made to the group allocation in order to ensure an acceptable distribution of the males to which females were mated.) Following allocation, the animals were earmarked by earpunch to give individual identification.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment.


BODY WEIGHT: Yes
- Time schedule for examinations:
All animals were weighed initially (=Day 1 of presumed pregnancy Batch A; Day 2 of presumed pregnancy Batch B) and on Days 2, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Bodyweights are reported from Day 2 of presumed pregnancy.

FOOD AND WATER CONSUMPTION: Yes
Food consumption was measured from weighday to weighday commencing on Day 3 of presumed pregnancy.
Gravimetric daily measurement of water consumption commenced on Day 3 of presumed pregnancy.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

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: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters, and results are presented in relevant tables of this report: bodyweight change, food and water consumption, litter data, sex ratio and foetal abnormalities and variants.

Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran 1967) followed by Williams' test (Williams 1971/2) or non-parametric tests (Kruskal-Wallis (Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977» were used to analyse these data, as appropriate. For litter data and foetal changes the basic sample unit was the litter and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used. Analyses of litter incidence of in utero deaths and foetal anomalies were performed using a trend test on the numberof litters affected, followed by a 2 sample permutation test (Edgington 1980; Franck 1986).

All significant (i.e. p≤ 0.05) intergroup differences from the control are reported only where supported by a significant analysis of variance (p ≤ 0.05).

Where 75% or more of the values for a given variable were the same, a Fisher's exact test (Fisher 1950) was used, when considered necessary.
Indices:
Individual litter values

In assessing litter parameters, pre-implantation loss was calculated as a percentage from the formula:

[(No. of corpora lutea - no. of implantations)/(No. of corpora lutea)] x 100

Post implantation loss was similarly calculated as a percentage from the formula:

[(No. of implantations - no. of live young)/(No. of implantations)] x 100

Litter weight and mean foetal weight were calculated from individual foetal weight.

Sex ratios at Day 20 were calculated as a percentage from the formula:

[(No. of live males)/(Total no. of live young)] x 100
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: Findings considered to be of no toxicological importance

Details on maternal toxic effects:
Clinical signs and mortalities
At 500 mg/kg/day, post-dosing salivation and associated wet coats were observed for all 25 animals during the treatment period. Post-dosing salivation was first observed on the second day of dosing. However, the number of rats affected on any particular day varied and was generally higher during the last four daysof treatment. Five animals also showed noisy respiration on isolated occasions between Days 11 and 15 of pregnancy.

At 125 mg/kg/day, 8/25 animals showed post-dosing salivation on isolated occasions during the last three days of the treatment period. One animal showed noisy respiration on Day 13 of pregnancy; since a higher incidence of noisy respiration was apparent at 500 mg/kg/day, this single incidence is considered
to be related to treatment.

At 30 mg/kg/day, one animal showed noisy respiration on Day 12 of pregnancy.

There were no deaths on the study.

Bodyweight change
At 500 mg/kg/day, treatment was associated with a significantly lower bodyweight gain during the first two days of treatment compared with the concurrent control. Thereafter, bodyweight gains to termination were generally comparable to controls.

At 30 and 125 mg/kg/day, there was no obvious adverse effect of treatment on bodyweight gain during pregnancy.

Food consumption
At 500 mg/kg/day, treatment was associated with lower food consumption during the first two days of treatment (Days 6 and 7) compared with the concurren control. Thereafter, food intake to termination was essentially comparable to controls.

At 30 and 125 mg/kg/day, there was no obvious adverse effect of treatment on food consumption. (It was noted that at 125 mg/kg/day, mean food intake during the first two days of treatment was significantly lower than in the control. However, as in the control group mean intake during this period was the same as that recorded prior to the commencement of treatment, the difference was considered to be of no toxicological importance).

Water consumption
At 500 mg/kg/day, water consumption was higher than in controls throughout the treatment period, differences from control attaining statistical significance for Days 8 to 15. This effect persisted, to termination, with differences from the control continuing to show statistical significance.

At 30 and 125 mg/kg/day, there was no obvious effect of treatment on water intake. (It was noted that at 125 mg/kg/day, water intake was significantly higher than in controls during Days 10 to 15. However, since differences from control values were only marginally greater than the difference recorded prior to the commencement of treatment, this finding is considered to be of no toxicological importance).

Macroscopic post mortem examination
The low incidence of occasional macroscopic changes at autopsy did not indicate any obvious adverse effect of treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Finding considered to be of no toxicological importance.

Details on embryotoxic / teratogenic effects:
LITTER DATA
There were 25, 23, 25 and 25 dams with live young at Day 20 sacrifice in Groups 1 to 4 respectively. Two dams in Group 2 were not pregnant.

There were no instances of total litter loss in utero.

There were no obvious adverse effects of treatment on the implantation rate, live litter size, sex ratio, litter weight, mean foetal weight or gravid uterine weight.

The incidence of early embryonic deaths in all treated groups was higher than in controls, with the litter distribution of early deaths (and consequently, total deaths) at 500 mg/kg/day being significantly different to that in the control group. However, since there was no dosage-relationship and, no instances of total litter loss in utero in treated groups (regardless of size no litter contained more than 3 dead embryos), the differences were considered likely to be coincidental and unrelated to treatment.

DETAILED EXAMINATION OF FOETAL MORPHOLOGY
The incidence of malfonned foetuses was 3/343, 0/296, 1/338, 11330, (2/25, 0123, 1125 and 1125 litters affected) foetuses examined in Groups 1 to 4 respectively. Neither the incidence, type nor distribution of malformations indicated any adverse effect of treatment.

There were no obvious effects of treatment on the number or distribution of litters or foetuses with either visceral or skeletal anomalies or, on the incidence of foetuses with skeletal variants.

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

Introduction

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.

The following guidelines were taken into consideration in the design and conduct of this study:

- OECD: Guideline for testing of chemicals No. 414, adopted May 1981.

- EEC: Council recommendation (83/571/EEC) Annex 1 - Repeated Dose Toxicity Official Journal of the European Communities L332, 20 - 22 October 1983.

- USA EPA: TSCA 798.4900 Development Toxicity Study, 20 May 1987.

- JMAFF: Requirements for Safety Evaluation of Agricultural Chemicals published in NohSan No. 4200, Ministry of Agriculture, Forestry and Fisheries 28 January 1985.

In this assessment of the effect of N-(n-hutyl) thiophosphoric triamide (NBPT), a urease inhibitor, on the pregnant rat and developing conceptus, dosages of 0 (Control), 30, 125 and 500 mg/kg/day were administered by intragastric intubation to groups of 25 time-mated females from Day 6 through to Day 15 of pregnancy. On Day 20 of pregnancy, the females were killed and subjected to post mortem examination, litter values determined and foetuses subsequently examined for visceral or skeletal changes.

Results

The following comments in relation to principal findings during the study are made in summary:

Treatment at 500 mg/kg/day was associated with significantly lower bodyweight gain and food consumption during the first two days of treatment compared with controls. Water consumption was significantly higher than in controls from Day 8 of pregnancy through to termination. All animals showed post-dosing salivation and associated wet coats and 5/25 animals showed noisy respiration. There were no obvious adverse effects on in utero development of the conceptus.

At 125 mg/kg/day, 8/25 animals showed post-dosing salivation and one animal showed noisy respiration. There was no indication of any adverse effect on in utero development of the conceptus.

At 30 mg/kg/day, one animal showed noisy respiration. There was no indication of any adverse effect on in utero development of the conceptus.

Conclusion

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.