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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between June 1996 - September 1996.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Details on test material:
Sponsor's identification : N-(n-butyl) thiophosphoric triamide (NBPT)
Description : Light tan waxy solid
Batch number : 8124 -165
Purity: 89.5% - 89.6%
Date received : 21 February 1996
Storage conditions : approx. 4ºC in the dark
Stability of test substance: December 1996

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston Road, Margate, Kent, England.
- Age at study initiation: At least 6 weeks
- Weight: 240 g to 283 g for males and 162 g to 207 g for females.
- Housing: The animals were placed at random in suspended cages with wire mesh floors so that each cage contained one rat. Each cage measured 21 cm wide, 26 cm deep and 27 cm high.
- Diet (e.g. ad libitum): Free access to food (pelleted diet).
- Water (e.g. ad libitum): Free access to mains drinking water.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature controls set to achieve target value of 21 ± 2ºC (Recorded values were generally within 21°C ± 3°C).
- Humidity (%): Relative humidity controls set to achieve target values of 55 ± 10% (Recorded values were generally 51 % ± 9% )
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to give 12 hours continuous light and 12 hours continuous dark per 24 hours.

IN-LIFE DATES: From: 10th June 1996 To: 12th September 1996

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: admixture with the diet
Details on oral exposure:
PREPARATION OF FORMULATIONS
The particle size of the test material was reduced prior to weighing by use ofa coffee grinder, after particle size reduction by hand if necessary. The test material was then accurately weighed out and plain diet was added, with stirring, to the ground material and the mixture was then ground using a coffee grinder. Dependent upon the concentration, a further amount of diet was added and the mixture ground again using a coffee grinder and diluted to the premix weight with further quantities of plain diet. Mixing of the premix was achieved using a Turbula mixer for a minimum of 5 minutes.

The dietary concentrations required for feeding were made by diluting 2000 g of the appropriate premix to the weight of the diet mix with plain diet, mixing being achieved using a Turbula mixer for a minimum of 5 minutes.

- Storage temperature of food:
Each diet mix was made from an individual premix. Diets were frozen until used.

DIETARY SAMPLING AND ANALYSIS
Prior to the start of treatment the proposed diet mixing procedures were checked by chemical analysis of trial diets to confirm that the proposed procedures produced homogenous diet, that the accuracy of mixing was acceptable and that the concentration of test substance in the diet remained unchanged between preparation and administration.

Samples of diets prepared for Week 1 and for Week 11 were also analysed to check the accuracy of preparation.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATIONS IN TEST DIET FORMULATIONS
At specified intervals (Weeks 1 and 11) during the study, freshly-prepared test diet formulations were sampled (200 g) from the turbula mixer drum by Pharmacy personnel and submitted for analysis. Each diet sample was sub-saInpled (10 g) in duplicate and analysed in accordance with Huntingdon Life Sciences analytical procedure IMF/FA/M112/95.

DETERMINATION OF THE TEST SUBSTANCE PURITY
The purity of the test substance (technical grade) was determined by HPLC assay prior to initiation and on completion of the neurotoxicity study.

At each occasion triplicate samples (approximately 50 mg) of the technical standard were prepared at a concentration of 12.5 µg/l in mobile phase and analysed in accordance with the analytical procedure with reference to the analytical grade standard.

The mean result for the purity of the technical grade N-(n-butyl) thiophosphoric triamide was confirmed as 89.5% - 89.6% for batch 8124-165.

The results for the test diet formulations analysed during the study were within 4% of nominal concentrations confirming the accuracy of formulation.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
The test substance, NBPT, was administered by admixture with the diet on a daily basis.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 1000 and 5000 ppm
Basis:
other: admixture with the diet
No. of animals per sex per dose:
Three groups of 15 males and 15 females per dose group for 0, 200, 1000 and 5000 ppm.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The treatment levels employed 0 (control), 200, 1000 and 5000 ppm were selected by the Sponsor based on a two week palatability/toxicity study performed in these laboratories.

- Rationale for animal assignment (if not random):
Random

Positive control:
No.

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS AND MEASUREMENTS

CLINICAL Signs and Mortality:
Individual animals were observed and palpated at least once daily for any signs of behavioural changes, reaction to treatment or ill health. These examinations were performed on each weekday, at suitable intervals during the day, for the first 4 weeks of the study and were not performed on Saturdays and Sundays. After 4 weeks of treatment, these examinations were performed once weekly.

Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. On Saturdays and Sundays and Public Holidays a similar procedure was followed except that the final checks were carried out at approximately mid-day.

There was a single mortality on this study following blood sampling in Week 5. A detailed macroscopic examination was performed and where practicable a full spectrum of tissue samples were preserved and examined.

Bodyweight
The weight of each rat was recorded at the time of allocation of animals to groups, on the day of commencement of treatment, and once a week thereafter.
Animals designated for the functional observational battery were also weighed on each occasion of testing.

Food consumption
The quantity of food consumed by each rat was recorded on a weekly basis for Week 1, the week prior to the start of treatment. Following the commencement of treatment, consumption was recorded on a daily basis. For the purposes of reponing, intake has been presented on a weekly basis.

Food intake per rat was calculated using the amount of food given to and left by each rat during each week using the following formula:

Food consumption (g/rat/week) = (Total food given - Total food left)/Number of animal days *

* The term 'animal day' counts one animal day for each animal alive for a whole day.

Efficiency of food utilisation

Efficiency of food utilisation (food conversion ratios) were calculated, where possible, on a weekly basis from Week 1 to 13 from the bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight. The following formula was used:

Food conversion ratio = Food consumed/Bodyweight gain

The 'food consumed' was calculated as indicated in the 'Food consumption' section and was not a mean of the individual cage means. The 'bodyweight gain' was calculated from the gain of each animal and used the mean gain in the formula.

Intake of test substance
At weekly intervals, the group mean achieved intake of test substance (mg/kg/day) was calculated from the group mean bodyweight and food consumption and the dietary inclusion levels of the test material. The following formula was used:

Achieved intake (mg/kg/day) = [Food consumed (g/rat/week) x ppm of test substance]/[Mid-week bodyweight (g/rat) x 7 (days/week)]

The 'food consumed' was calculated as indicated in the Food consumption section and was not a mean of the individual cage means. The mid-week bodyweight was calculated from each individual animal and used the mid-week bodyweight in the formula.

Water consumption
Daily monitoring by visual appraisal of the water bottles was maintained throughout the study.

Water consumption was measured accurately, by weight, over daily periods during Week 12 for all rats in all groups.

Ophthalmic examination
Before treatment commenced, the eyes of all animals were examined by means of a Keeler indirect ophthalmoscope. During Week 13 the eyes of all animals in Groups 1 and 4 were examined.

Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

The ophthalmoscopic examination performed prior to the commencement of treatment resulted in the rejection of one male and 5 female rats:

- one male with right eye prominent hyaloid remnant and small circular opacity;
- one female with right eye a patch of hyper-reflectivity in fundus;
- one female with bilateral retinal disruption around disc;
- one female with left eye intravitreal haemorrhage;
- one female with right eye posterior capsular polar opacity;
- one female with right eye pupil undilated after re-dropping with Mydriacil.


Neurotoxicity screening (Main and Satellite groups)
There were transitory changes in behaviour during Week 4 which were characterised by decreased grip strength for all animals treated at 5000 ppm and a low incidence of hunched posture in females at 5000 ppm. With continuing exposure, these changes disappeared.

The functional observational battery and motor activity test were performed on a total of 10 animals/sex/group (5 Main group animals/sex/group and 5 Satellite animals/sex/group).

During the study, a functional observational battery and motor activity were performed at approximately the same time of day, before initiation of treatment and during the 4th, 8th and 13th week of treatment. Not all rats were tested in one day, but time of testing was balanced across the groups. Observations made during the treatment period were made in the afternoon. In addition, observations in Week 13 were performed prior to any laboratory investigations.

The functional observational battery is detailed below:
The battery comprised 4 sets of observations. The first set of observations was performed while the animal was in its home cage. The second set was performed when initially handling the animal. The third set of observations was performed in the test arena and the fourth set comprised handling/specific testing of the animal. All these observations were made with the observer blind to the treatment condition of the animal.

Horne cage observations:
Posture in the cage
Presence of convulsions, tremors, twitches
Presence of spontaneous vocalisations
Palpebral closure

Observations in the hand:
Ease of removing the animal from the cage
Ease of handling the animal
Salivation/lacrimation
Palpebral closure
Exophthalmus
Piloerection
Vocalisation on handling

Observation in the arena:
Occurrence of convulsions, tremors, twitches
Level of activity in the arena (number of squares entered counted)
Level of arousal
Rearing count
Grooming
Assessment of gait
Record presences of faecal boluses, urine

Manipulations:
Approach response
Touch response
Startle response
Righting reflex
Tail pinch response
Pupil reflex
Grip strength (fore and hindlimb)
Landing foot splay
Body temperature (⁰C)
Bodyweight (g)

At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity was performed and monitored using a Coulbourn Infra-Red Activity Monitoring System (system supplied by Coulbourn Instruments, Lehigh Valley, PA, USA).

This system uses an infra-red detector to monitor activity. The following categories of activity are recorded: the time spent in no movement, locomotor, and non-locomotor activity. The number ofoccurrences (events) of each category is also recorded. For reporting this data, only the time spent in
locomotor activity was presented.

For testing, designated animals were placed singly into observation cages. Once all animals had been placed into the cages, the test session programme was started. The test session for each animal was 1 hour. Data was collected every 2 minutes and written onto a floppy disk.

LABORATORY INVESTIGATIONS (Main groups)

During Weeks 5 and 13, samples of blood were withdrawn, under light anaesthetic, from the orbital sinus of all rats from each group.

The blood samples collected were divided into tubes as follows:
EDTA anticoagulant..................................... for haematological investigations
Citrate anticoagulant................................... for coagulation tests
Heparin anticoagulant . . . . . . . . . . . . . for biochemical tests

Food was removed overnight from animals sampled for laboratory investigations.

The estimations performed on blood samples have been listed below , together with an abbreviated title the methods and the units of measurement applicable at the time.

Haematology
The following estimations were performed using a Bayer-Technicon H1E haematology analyser:
Packed cell volume (PCV)....................................................................................................................%
Haemoglobin (Hb)...............................................................................................................................g/dl
Red blood cell count (RBC)..............................................................................................................x 10E12/l
Absolute indices were calculated as follows:
Mean corpuscular haemoglobin concentration (MCHC) Hb (g/dl) x 100 + PCV (%).............g/dl
Mean corpuscular volume (MCV) PCV (%) x 10 ÷ RBC (x 10E6/mm3)......................................fl
Mean corpuscular haemoglobin (MCH) Hb (g/dl) x 10 ÷ RBC (x I0E12/l)...............................pg
Total white cell count (WBC total).....................................................................................................x10E9/l

Differential WBC count (Diff):
- Neutrophils (N)
- Lymphocytes (L)
- Eosinophils (E)...............................................................................x 10E9/l
- Basophils (B)
- Monocytes (M)
- Large unstained cells (LUC)

Cell morphology: the most common morphological changes (anisocytosis, micromacrocytosis, variation in colour, hypo/hyperchromasia, left shift, atypical blast cells)
were recorded as follows:
- = no abnormalities detected
+ = slight
++ = moderate
+++ = marked
In the case of atypical blast cells, or other abnormalities, confirmation or a written description from a blood film was made.

Platelet count (PIts)................................................................................x 10E9/l
Thrombotest (TT) - Owren, P.A. (Lancet, 1959, ii, 754)..................s

Biochemistry
The following parameters were analysed with a Hitachi 737 Clinical Chemistry Analyser:
Total protein (Protein Total)...................................................................................................................g/dl
Albumin (Alb)............................................................................................................................................g/dl

Globulin (Glob) - by subtraction
Total protein (g/dl) minus Albumin (g/dl)............................................................................................g/dl
Urea nitrogen (Urea Nitr).........................................................................................................................mg/dl
Creatinine...................................................................................................................................................mg/dl
Sodium (Na)................................................................................................................................................mEq/l
Potassium (K).............................................................................................................................................mEq/l
Calcium (Ca)...............................................................................................................................................mEq/l
Inorganic Phosphorus (P)........................................................................................................................mEq/l
Chloride (Cl)...............................................................................................................................................mEq/l
Cholesterol (Chol) - (enzymatic assay).................................................................................................mg/dl

Alkaline phosphatase (AP)
Reaction temperature 30°C......................................................................................................................mU/ml

Total bilirubin.............................................................................................................................................mg/dl
Glucose (Hexokinase mediated assay)...................................................................................................mg/dl

Glutamic-pyruvic transaminase (GPT), also known
as 'alanine aminotransferase'
Reaction temperature 30°C......................................................................................................................mU/ml

Glutamic-oxaloacetic transaminase (GOT), also known
as 'aspartate aminotransferase'
Reaction temperature 30°C.......................................................................................................................mU/ml

yGlutamyl transferase (yGT)
Reaction temperature 30°C.......................................................................................................................mU/ml

Ornithine carbamoyl transferase (OCT)
Reaction temperature 37 °C......................................................................................................................mU/ml



Sacrifice and pathology:
GROSS PATHOLOGY: Yes

TERMINAL STUDIES (Main groups)
On completion of 13 weeks of treatment, all surviving rats were killed.

As the terminal procedures took several days to complete, individual animals continued to receive the appropriate control or test diet until the day on which they were killed. The duration of the dosing period, however, was reported as 13 weeks.

All rats were killed by carbon dioxide asphyxiation and subjected to the necropsy procedure indicated below :

- All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera were noted with due attention to the thymus, lymph nodes and heart.

- The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighed:adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus. Testes and epididymides were weighed individually and identified as left or right. The weights of major organs of individual rats dying or killed during the study were recorded at the discretion of the pathologist.


HISTOPATHOLOGY: Yes

Histopathological examination

Tissues required for microscopic examination in this study are marked '*' in the list below.

adrenals, alimentary tract, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, aorta, brain (medullary, cerebellar and cerebral sections), epididymides, eyes, femur (with joint), Harderian gland, head (to preserve nasal, cavity, paranasal sinuses,, oral cavity, nasopharynx, teeth,, lachrymal gland and, Zymbal's gland), heart, kidneys, larynx and pharynx, liver, lungs (all lobes and mainstem bronchi) lymph nodes (cervical and, mesenteric), mammary gland, ovaries, other macroscopically, abnormal tissue, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin spinal column (to preserve samples of spinal cord from cervical level), spleen sternum (for bone and marrow), testes, thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus (corpus and cervix), vagina.

For testes and epididymides, tissues were embedded in paraffin wax and sections were stained with PAShaematoxylin. A transverse section of each testis and a full longitudinal section of each epididymis was cut as near as possible to 2 micrometres. Microscopic assessment of the testes was made with reference to the stages of the cycle of the seminiferous epithelium. Other tissues were embedded in paraffin wax and sections cut at 4 micrometres were stained with haematoxylin and eosin.

Frozen sections of liver, fixed in buffered formalin, were cut on a cryostat at 12 micrometres and stained for fat with Oil Red O (ORO). Sections of kidney were stained with ORO or Periodic acid-Schiff reagent (PAS) at the discretion of the pathologist.

Histopathological examinations were restricted to:

-The specified list of tissues including all macroscopically abnormal tissues from all animals from the control group, and all animals from the high dosage level group, whether dying or killed at 13 weeks.

-Any macroscopically abnormal tissue in any animal.
-Lungs, liver and kidneys from all rats in the low and intermediate dosage level groups.


TERMINAL STUDIES (Satellite animals)

On completion of 13 weeks of treatment, Satellite group animals were killed.

All animals were perfused and tissue samples were taken from them, but neuropathological examination was restricted to the control and high dose groups.

Method of sacrifice and perfusion fixation: the animals were anaesthetised with sodium pentobarbital (ip) and perfused in situ with heparinised 0.7 % sodium nitrite followed by a 1.5 glutaraldehyde: 4 % paraformaldehyde solution. After perfusion,the cranial vault was removed and the brain removed,
weighed and measured as detailed below. The skin was removed from the dorsal region and hindlimbs, and the sciatic, tibial and sural nerves were exposed. The spinal column and cord was fixed in toto integral with the carcass overnight in fixative-filled containers and held at 4°C.

Anatomical measurements: the brian was transected from the spinal cord above the first cervical spinal nerve, and the olfactory lobes were removed. The length was measured between the rostral part of the cerebral hemispheres to the most caudal part of the cerebellum, and the width between the widest parts of the cerebral hemispheres.

Following overnight storage, tissues for each animal for paraffin wax H&E sections were taken and stored for at least 48 hours in buffered formalin. Peripheral nerve samples were taken and processed for epon/toluidine blue sections. The carcass was transferred to buffered formalin and stored at room temperature.

Light microscopic examination: brain, spinal cord, ganglia and dorsal and ventral root fibres of the perfused animals were processed for paraffin embedding, sectioned at approximately 5 - 6 microns and stained with haematoxylin and eosin. Peripheral nerves from the right side were embedded in epon, sectioned at approximately 2 microns, and stained with toluidine blue.

Other examinations:
None.
Statistics:
All statistical analyses were carried out separately for males and females.

For all parameters the analyses were carried out using the individual animal as the basic experimental unit.

The following data were routinely subjected to statistical analysis: rearing and activity counts, grip strength, hindlimb splay, bodyweight and temperature. These data were analysed using a one-way analysis of variance followed by Williams' test (Williams 1971/2) for a dose-related response. Pre-dose data was analysed by analysis of variance followed by Student's t test. The reporting of the categorical data for the observational battery has been handled in the following
manner. The observational endpoints such as ease of handling, arousal, etc have been tabulated for frequency of occurrence for each group. Although during recording, some responses were classified in tenns of the degree or type of response (ie startle: no reaction, an ear twitch, a flinch, etc), for the purposes of reporting, as there were no remarkable differences between the groups, for a given endpoint the response has been reported as being either present or absent.

The categorical data were analysed in the following manner:
Where the recorded observations suggested a possible difference between controls and treated groups, the data were analysed using the Linear by Linear Association test (Agresti, 1990; Agresti, Mehta and Patel, 1990). The data for arousal was analysed using Jonckheere-Terpstra test (Hollander & Wolfe, 1973; StatXact, 1992). All the recorded categories were used in the analysis. A two tailed test was generally reported except where the responses were considered directional
in nature.
The Coulbourn activity data were analysed using a one-way analysis of variance followed by Williams' test (Williams 1971/2) for a dose-related response. Pre-dose data were analysed by analysis of variance followed by Student's t test.


Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Mortality:
mortality observed, treatment-related
Description (incidence):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No statististically significant difference compared with with controls.The toxicological significance of the findings, if any, cannot be determined at this time.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The only change noted at macroscopic examination at terminal autopsy was uterine fluid distension in 0, 0, 5, 6 females in Groups 1 to 4 respectively. There were no other macroscopic changes which were considered related to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No abnormalities detected
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No abnormalities detected
Details on results:
FORMULATION ANALYSIS
The results of the analysis of the diet samples from Weeks 1 and 11 were in good agreement with the intended nominal concentrations with the results being within 4% of nominal concentration.

CLINICAL SIGNS AND MORTALITIES
The only clinical signs considered associated with treatment were limited to one female being treated at 5000 ppm. This animal was observed as having a range of signs including unsteady gait, hunched posture and piloerection. All these signs had disappeared by Week 10 of the study.

There was a higher incidence of males treated at 5000 ppm with crooked teeth (2, 1, 2 and 5 in Groups 1 to 4 respectively). However, this was considered to be coincidental and not of toxicological importance.

There was one mortality, during the course of the Week 5 blood sampling procedure, a control female failed to recover from the anaesthetic.

BODYWEIGHTS
Bodyweight gains among males and females treated at 5000 ppm were lower than controls from the commencement of treatment with the cumulative gains from Week 0 to 4 being statistically significantly lower than controls. During Weeks 4/5, gains for both males and females became generally comparable with controls. The gains for females during Weeks 4 to 8 were statistically significantly increased compared with controls. Towards the end of the study (Weeks 8 to 12), there was a suggestion among males at 5000 ppm of a further slight reduction in weight gains.

At lower dosages, the pattern of weight gains was comparable with controls.

FOOD CONSUMPTION
Cumulative food intakes were statistically significantly lower among males at 5000 ppm compared with controls. There was no similar effect among females, although it was noted that during the first few weeks of treatment intake was slightly lower when compared with controls.

At lower dosages, intake was comparable with controls.

EFFICIENCY OF FOOD UTILISATION
Treatment at 5000 ppm was initially associated with an impairment of food utilisation. Among males, there was evidence of increasing impairment from the commencement of treatment through to Week 3. Among females, there was a very marked impairment during Weeks 2 and 3. From Week 4 onwards, food utilisation showed a complete recovery with values being comparable to or better than controls.

At lower dosages, values were considered comparable with controls.

ACHIEVED MEAN INTAKE
The achieved mean intake of test material was as expected in a fixed inclusion level study. There was no overlap between the selected dosages and generally there was a five-fold interval in the achieved intakes.

The overall achieved intake in terms of mg/kg/day was 14.7, 74, 377 for Main group males and 17.4, 88 and 445 for Main group females.

WATER CONSUMPTION
There were no statistically significant differences in water consumption between treated and control groups.

OPHTHALMOSCOPY
The ophthalmoscopic findings were within normal limits for animals of this age and strain. None of the changes observed were considered related to treatment.

NEUROBEHAVIOURAL OBSERVATIONS

Observational endpoints:

Emaciated appearance was recorded on all occasions with statistically significant differences in the incidence of observations occurring during Week 4 and 8 for both sexes treated at 5000 ppm and for females only during Week 13.

During Week 4, hunched posture was observed in two females at 5000 ppm. The observation of grooming while in the arena was statistically significantly higher among males treated at 5000 ppm compared with controls, however, no toxicological importance is attached to this singular observation.

Among males treated at 1000 or 5000 ppm, there was a statistically significant increase in the numbers of animals vocalising on removal from the cage compared with controls during Week 8. During Week 13, although there was no dosage relationship, all treated males were more likely to vocalise compared with controls.

Among males treated at 5000 ppm, there was a slight shift to a lower level of arousal compared with controls. This was first observed in Week 8. It was still present in Week 13 with differences from control just failing to attain statistical significance.

There was an increase in the incidence of males at 5000 ppm for which assessment of gait was not possible due to limited movement in the arena. This was observed in Week 8 and 13 with the incidence attaining statistical significance in Week 13.

During Week 13, there was a statistically significant increase in the incidence of slight eye closure in the arena among males treated at 5000 ppm.

Singular observations made during the course of the study but which could not be clearly attributed to
treatment included the following:
• cold feet: Week 4 one male at 200 ppm and one male at 1000 ppm; Week 8 one female at 200 ppm;

• firm abdomen: Week 4 one male at 5000 ppm, Week 8 one male at 1000 ppm

• slightly swollen abdomen: Week 8 one female at 5000 ppm

• rapid breathing: Week 4 one male at 5000 ppm.


Rearing and activity counts
There were no statistically significant differences in rearing or activity counts between treated and control groups throughout the treatment period.

Grip strength
During Week 4, there were statistically significant lower values recorded for both forelimb and hindlimb grip strength among males and females treated at 5000 ppm. During Weeks 8 and 13 no statistically significant differences in grip strength, although it was noted in Week 8 that females treated at 5000 ppm did show slightly lower mean grip strength values when compared with controls.

At lower dosages, values were comparable with controls throughout treatment.

Hind limb splay
There were no statistically significant differences in mean splay values between treated and control groups throughout the treatment period.

Rectal temperature
There were no statistically significant differences in mean rectal temperature between treated and controls throughout the treatment period.

Bodyweight
During Week 4, statistically significant lower mean bodyweights were recorded for males and females treated at 5000 ppm compared with their respective controls. At lower dosages, bodyweights were comparable with controls. During Week 8 and 13, statistically significant lower mean bodyweights were recorded for males treated at 5000 ppm compared with controls. During Week 8 and 13, among females at 5000 ppm, bodyweights remained lower than controls but differences failed to attain statistical significance.

At lower dosages, there were no statistically significant differences from controls.

Coulbourn activity monitoring
There were no statistically significant differences in activity among treated and controls animals throughout the treatment period.

Discussion
Treatment with NBPT for thirteen weeks was associated with some behavioural changes as well as clear evidence of toxicity as evidenced by effects on bodyweight. The behavioural changes and evidence of toxicity were mainly confined to treatment at the highest dosage, 5000 ppm. The observations appeared to change during the course of exposure.

During Week 4, there were clear effects on bodyweight (lower weights) and an observation in some animals at 5000 ppm of emaciated appearance. There was a statistically significant lower grip strength values for both males and females at 5000 ppm. This may partly reflect the effect on bodyweight, however, the degree of the effect on hindlimb grip strength among females at 5000 ppm could not solely be related to bodyweight as the bodyweight at Week 4 was greater than in the predose period and yet the grip strength in Week 4 was lower than the predose period. At later weeks, although bodyweights continued to be lower than controls, grip strength values recovered to control levels and there were no statistically significant differences from controls. Other changes in Week 4 included a low incidence of hunched posture among females at 5000 ppm.

During Week 8 and 13, there was a tendency for more treated males to vocalise when being handled compared with controls. This was interpreted as possibly a non-specific effect of treatment.

During Week 13, among males treated at 5000 ppm, there were observations of slight eye closure in the arena, an increase in the number of animals for which assessment of gait was not possible and a tendency for a lower level of arousal. These signs of slight eye closure and lack of gait assessment were considered to be related to the lower arousal evidence these animals. With lower arousal, the expectation would be for lower activity and rearing counts, however, there were no statistically significant effects on either behaviour nor was there any significant effect in activity in the Coulboum activity system. In the absence of changes in other behaviours, the changes in arousal and associated effects are therefore considered to be of doubtful toxicological significance.

To conclude, treatment with NBPT for thirteen weeks was associated with a transitory change in behaviour during Week 4 which was characterised by decreased grip strength for all animals treated at 5000 ppm and a low incidence of hunched posture in females at 5000 ppm. With continuing exposure, these changes disappeared.

HAEMATOLOGY

Week 5
Among males at 5000 ppm, the total white blood cell counts were statistically lower than controls. This was attributed to the statistically significantly lower lymphocyte counts at 5000 ppm. Among females, there was a statistically significant increase in platelet counts at 5000 ppm.

Week 13
Among females at 5000 ppm, there was a statistically significant increase in platelet counts at 5000 ppm.
Other statistically significant differences from control values were observed in Weeks 5 and 13 but were considered to be marginal in nature and of no toxicological importance.

BIOCHEMISTRY

Week 5
Among males, GPT values were statistically significantly decreased at 5000 ppm, and GOT values were statistically significantly decreased at 1000 and5000 ppm. Phosphorus levels were also statistically significantly lower at 5000 ppm.

Among females, AP, GPT and GOT values were statistically significantly decreased at 5000 ppm compared with controls.

Week 13
Among males, AP values were statistically significantly decreased at 1000 and 5000 ppm. GOT values were statistically significantly decreased at 5000ppm. Phosphorus levels were also marginally, but statistically significantly lower at 5000 ppm.

Among females, GPT values were statistically significantly decreased at 5000 ppm compared with controls. (Although mean GPT values at 1000 ppm, were also lower than controls, the values were highly variable and failed to attain statistical significance.)

Among females phosphorus levels were statistically significantly decreased at all dosages of NBPT. It was noted that there was a wide range of values for the controls. Examination of background data showed a 95% range of 2.4 - 4.3 with a mean of 3.3. Values for females treated at 200 and 1000 ppm were therefore within background.

Other statistically significant differences from control values were observed in Weeks 5 and 13 but were considered to be marginal in nature and of doubtful toxicological significance.

ORGAN WEIGHTS
Among males at 5000 ppm, there was a statistically significant increase in liver weights when adjusted for bodyweights. Among females, there was a statistically significant increase in uterus weights compared with controls at 1000 and 5000 ppm.

MACROSCOPIC PATHOLOGY
The only change noted at macroscopic examination at terminal autopsy was uterine fluid distension in 0, 0, 5, 6 females in Groups 1 to 4 respectively.There were no other macroscopic changes which were considered related to treatment.

BRAIN MEASUREMENTS
There were no statistically significant differences from controls in any of the brain measurements.

MICROSCOPIC PATHOLOGY

Terminal kill - Main group animals - toxicity screen:
Treatment-related changes

Liver - Minimal centrilobular hepatocyte hypertrophy was reported in male rats at 1000 and 5000 ppm.

MALE
Dosage level (ppm) 0 200 1000 5000
Minimal centrilobular
hepatocyte hypertrophy 0 0 2 6**
Number livers examined 10 10 10 10

** p < 0.01 with Fisher's Exact Test


This finding attained statistical significance in male rats at 5000 ppm and was considered to be treatmentrelated in this group. The incidence reportedin male rats at 1000 ppm was within the limits of background histopathological findings reported in this age and strain of laboratory-maintained rats.

Minimal centrilobular hepatocyte hypertrophy was reported in a proportion of control and treated female rats but was not considered to be treatment-related.

Uterus - Luminal dilatation was reported in all treated groups of female rats.


FEMALE
Dosage level (ppm) 0 200 1000 5000
Luminal dilatation 0 3 5 6
Number uteri examined 9 3 5 10

This finding showed a dose-related incidence and was considered to be treatment-related in all treated groups of rats.

Although fluid distension of the uterus is a normal physiological event associated with oestrus cycling, the significance of its occurrence in treated groups is unknown.


Kidneys - Foci of dystrophic mineralisation were reported in the kidneys in a proportion of control and treated female rats.


FEMALE
Dosage level (ppm) 0 200 1000 5000
Dystrophic mineralisation
Total 2 2 2 6
Minimal 2 2 1 3
Moderate 0 0 1 3
Number kidneys examined 9 10 10 10

Although this finding did not attain statistical significance in any group there was an increased incidence and severity of mineralisation in the 5000 ppm group which may be related to treatment. Foci of mineralisation in the kidneys of female rats is a sex-related spontaneous finding considered to be related to circulating oestrogen levels. In view of the uterine findings in treated females, it is possible that the increase in renal mineralisation at 5000 ppm may reflect an altered hormonal balance.

Terminal kill - Satellite animals -neurotoxicity screen

No treatment-related effects were reported in any tissues available for histopathological examination.

Conclusion
Terminal kill main group animals - Treatment-related findings were reported in the liver, uterus and kidneys.

Liver - Centrilobular hepatocyte hypertrophy in male rats receiving 5000 ppm.
Uterus - Luminal dilatation in all treated groups of rats.
Kidneys - Foci of mineralisation in female rats receiving 5000 ppm.

Terminal kill satellite animals - No treatment-related findings were reported.

Effect levels

Dose descriptor:
NOEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION AND CONCLUSION

Treatment with the test substance for thirteen weeks at dietary inclusion levels of 0, 200, 1000 and 5000 ppm was associated with a range of effects principally at 5000 ppm.

For some of the observations at 5000 ppm, the pattern of reaction to treatment changed with continuing exposure such that an initial reaction was foHowed by recovery. This included a single female which showed clinical signs up to Week 10 of the study; an initial reduction in bodyweight gains, an impairment in food utilisation for the first 4 weeks of treatment, and observations on the functional observational battery when performed in Week 4 but not at subsequent intervals. Other observations showed a continuing effect such as the lower cumulative food consumption among males at 5000 ppm.

Treatment at 5000 ppm was also associated with effects on some haematology, and biochemistry parameters. Organ weight changes were limited to increased liver weights for males and increased uterine weights for females. The increased liver weights was associated with centrilobular hepatocyte hypertrophy and the increased uterine weight was associated with luminal dilatation of the uterus. There was also an increase in the incidence of foci of mineralisation in the kidney among females.

The behavioural assessment showed transitory changes in Week 4 at 5000 ppm but there were no subsequent changes. Neuropathology after 13 weeks of exposure provided no evidence of neurotoxicity of NBPT.

At 1000 ppm, effects were limited to changes in some biochemistry parameters in both males and females and effects on the uterus (luminal dilatation) among females.

At 200 ppm, effects were limited to females and included statistically significantly lower phosphorus levels (which was within background) and a low incidence of luminal dilatation of the uterus.

To conclude, treatment with NBPT for thirteen weeks was associated with clear signs of toxicity at 5000 ppm. At 1000 ppm, there were limited effects on some biochemistry parameters and among females, an increased incidence of luminal dilatation of the uterus.

The no observed effect level (NOEL) was established in the males as being 200 ppm. It was not possible to demonstrate a NOEL among females as a slightly lower phosphorus level and a low incidence of luminal dilatation of the uterus was observed. The tox~cological significance of these findings, if any. cannot be determined at this time.

Applicant's summary and conclusion

Conclusions:
Treatment with NBPT for thirteen weeks at dietary inclusion levels of 0, 200, 1000 and 5000 ppm was associated with a range of effects.

At 5000 ppm, treatment was associated with some reactions that showed subsequent recovery (clinical signs, bodyweight gains, food utilisation, and responses on the functional observational battery) as well as some effects that continued throughout treatment (cumulative food consumption and haematology and biochemistry parameters). Organ weight and histopathological changes were also observed.

At 1000 ppm, there were limited effects on some biochemistry parameters and among females, an increased incidence of luminal dilatation of the uterus.

The no observed effect level (NOEL) was established in the males as being 200 ppm. However, it was not possible to demonstrate a NOEL among females as a slightly lower phosphorus level and a low incidence of luminal dilatation of the uterus was observed. The toxicological significance of these findings, if any, cannot be determined at this time.
Executive summary:

Introduction.

The test substance was assessed for repeat oral dose toxicity.

The object of this study was to assess the toxicity and the potential neurotoxicity of the test substance, NBPT, to rats by dietary administration over a period of 13 weeks.

The neurotoxicity component was designed in accordance with EPA FIFRA Pesticide Assessment Guidelines, SubdivisionF, Addendum 10, Neurotoxicity, published March 1991, which is also applicable to neurotoxicity testing of industrial chemicals for TSCA. Otherwise, this study was designed in accordance with EPA TSCA Test Guidelines, 40 CFR Part 798 - Health Effects Testing Guidelines, September 27 1985, 798.2650, Oral Toxicity and Subsequent Revision, May 20 1987.

The treatment levels employed 0 (control), 200, 1000 and 5000 ppm were selected by the Sponsor based on a two week palatability/toxicity study performed in these laboratories (IMF/6).

The rat was the species of choice due to regulatory requirements and the Crl:CD BR strain was chosen due to availability of background data.

The route of exposure, dietary, was selected since the oral route is a potential route of human exposure to the test material.

Methods.

In this experiment, the toxicity of (N-(n-butyl) thiophosphoric triamide (NBPT), an organophosphorus compound and a urease inhibitor, was assessed in three groups of 15 male and 15 female Crl:CD BR rats which were given NBPT by dietary admixture at levels of 200, 1000 and 5000 ppm for 13 weeks. Dietary inclusion levels remained constant throughout the study. The overall achieved mean intakes in terms of mg/kg/day for main animals were 14.7, 74 and 377 for males and 17.4, 88 and 445 for females. A similar sized group was given untreated diet and acted as controls. Ten animals/sex/group were designated as Main group animals with the remaining five animals/sex/group designated as Satellite animals.

All animals were monitored for clinical signs, bodyweights and food consumption. Water consumption was measured during Week 12. The eyes of all control animals and animals treated at 5000 ppm were subjected to an ophthalmoscopic examination in Week 13. Laboratory investigations (haematology and clinical chemistry) were performed on all Main group animals in Weeks 5 and 13.

During the pre-dose period and Weeks 4, 8 and 13, 5 males and 5 females from the Main group and all the Satellite animals were screened for neurotoxicity (ie scored on a functional observational battery and were quantitatively assessed for locomotor activity). At Week 14 all the Satellite animals were killed using whole body perfusion and selected tissues of the nervous system were then examined histopathologically. At Week 14 all Main group animals were killed, a post mortem examination performed and organ weights were recorded for selected tissues. Histopathology was performed on indicated tissues.

Results.

Clinical signs and mortalities

One female treated at 5000 ppm was considered to be affected by treatment showing signs of unsteady gait, hunched posture and piloerection. These signs were exhibited during Weeks 3 to 10.

There were no mortalities associated with treatment.

Bodyweights

Cumulative gains were statistically significantly lower than controls among males and females treated at 5000 ppm from the commencement of treatment to Week 4. There was a suggestion among males at 5000 ppm of a slight reduction in weight gains during Weeks 8 to 12.

Food consumption

Cumulative intakes (Weeks 1 to 12) among males treated at 5000 ppm were statistically significantly lower than controls.

Efficiency of food utilisation

During the first 3 weeks of treatment, food utilisation was clearly impaired among males and females treated at 5000 ppm.

Water conswnption

There was no effect of treatment.

Neurotoxicity screening

There were transitory changes in behaviour during Week 4 which were characterised by decreased grip strength for all animals treated at 5000 ppm and a low incidence of hunched posture in females at 5000 ppm. With continuing exposure, these changes disappeared.

Ophthalmoscopic examination

There was no effect of treatment.

Haematology

During Week 5, white blood cell counts were statistically significantly lower among males treated at 5000 ppm due to increased lymphocyte counts.

Among females treated at 5000 ppm, platelet counts were statistically significantly increased at Week 5 and Week 13.

Biochemistry

Statistically significant differences were observed in the following parameters at 5000 ppm:

  • GPT decreased among males in Week 5 and among females in Weeks 5 and 13; GOT decreased among males in Weeks 5 and 13, and among females in Week 5; AP decreased among males in Week 13 and among females in Week 5; phosphorus levels decreased among males in Weeks 5 and 13 and among females in Week 13;

At 1000 ppm the following parameters were statistically significantly different:

  • GOT decreased among males in Week 5; AP decreased among males in Week 13; phosphorus levels decreased among females in Week 13;

At 200 ppm the following parameters were statistically significantly different:

  • Phosphorus levels decreased among females in Week 13;

Organ weights

Liver weights when adjusted for bodyweights were statistically significantly increased among males treated at 5000 ppm when compared with controls. Uterus weights of females treated at 1000 or 5000 ppm were statistically significantly increased compared with controls.

Macroscopic pathology

An increased incidence of fluid distension of the uterus was observed among females treated at 200, 1000 or 5000 ppm compared with controls.

Histopathology

Among males, a statistically significantly increased incidence of minimal centrilobular hepatocyte hypertrophy was observed in male rats 5000 ppm. Among female rats, at 5000 ppm, an increase in the incidence of foci of mineralisation in the kidney was observed. There was also a treatment related increase in the incidence of luminal dilatation of the uterus for females treated at all dosages of NBPT.

Neuropathology provided no evidence of neurotoxicity.

Conclusion

Treatment with NBPT for thirteen weeks at dietary inclusion levels of 0, 200, 1000 and 5000 ppm was associated with a range of effects.

At 5000 ppm, treatment was associated with some reactions that showed subsequent recovery (clinical signs, bodyweight gains, food utilisation, and responses on the functional observational battery) as well as some effects that continued throughout treatment (cumulative food consumption and haematology and biochemistry parameters). Organ weight and histopathological changes were also observed.

At 1000 ppm, there were limited effects on some biochemistry parameters and among females, an increased incidence of luminal dilatation of the uterus.

The no observed effect level (NOEL) was established in the males as being 200 ppm. However, it was not possible to demonstrate a NOEL among females as a slightly lower phosphorus level and a low incidence of luminal dilatation of the uterus was observed. The toxicological significance of these findings, if any, cannot be determined at this time.