N-butylphosphorothioic triamide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: December 4, 1995, Experimental Initiation Date: January 26, 1996; In-Life Experimental Completion Date: September 27, 1996; Metabolite 10 Experimental Completion Date: January 27,1997; Study Completion Date: 12 June 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deveiations from standard test guidelines and./or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): [14C]-N-(n-butyl)thiophosphoric triamide
- Lot number: 950404
- Stated radiochemical purity: 98.7%
- Specific activity: 25.12 mCi/mmol

AND

- Chemical name: N-(n-butyl)thiophosphoric triamide
- Lot number: 1-12-3
- Stated chemical purity: 99.2%
- Molecular weight: 167

The radiolabeled and nonradiolabeled test substances were supplied by the Sponsor and then stored at < -15 °C and room temperature, respectively. The Sponsor indicated that the test substances were characterized prior to study initiation under GLP.

The radiochemical purity of the [14C]-N-(n-butyl) thiophosphoric triamide was determined to be 94.8% prior to the preparation of the dosing materials.

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Sprague-Dawley CrI:CD VAF/Plus

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., 9801 Shaver Road, Portage, Michigan 49801.
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 209 to 234 g

- Fasting period before study:
12 to 16 hours prior to the administration of the test material. Food was returned to the animals 4 hours after dosing.

- Housing:
During acclimation, animals were housed in stainless steel cages. During both experiments, animals were housed individually in glass metabolism cages designed for the collection of expired air, urine and feces. The size of the cages met the standards recommended by the Guide for the Care and Use of Laboratory Animals.

- Individual metabolism cages: yes
- Diet: The animals were fed with Certified Rodent Laboratory Chow® No. 5002 ad libitum
- Water (e.g. ad libitum): Fresh potable water was provided ad libitum at all times during the acclimation and throughout the study.
- Acclimation period: At least 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 65 to 78 °F
- Humidity (%): 40 to 70%
- Air changes (per hr): At least 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): An alternating 12 hour light/dark cycle maintained using an automatic timing switch.

IN-LIFE DATES: FROM: Experimental Initiation Date: 26 January 1996 to In-Life Experimental Completion Date: 27 September 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose/water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
[14C]NBPT was isotopically diluted with [12C]NBPT. The stock solution of [14C]NBPT in methanol was added to solid [12C]NBPT. The solvent was evaporated completely from the solution using a stream of argon gas at ambient temperature. The test material was then suspended in 0.75% (w/v) methylcellulose (400 centipoise)/HPLC grade water.

In the pilot experiment, chemical and radiochemical concentrations of the dose preparation were determined prior to and after dosing by HPLC and
LSC. The specific activity of the dose preparation and the dose amount administered to each animal (mg or J.1Ci) were determined based on the analytical data.

In the main experiment, only the radiochemical concentration of the dose preparation was determined prior to and after dosing. Nominal chemical concentration and specific activity of the dose preparation were used to calculate the specific activity of the dose preparation and the dose amount administered to each animal. The nominal chemical concentration and specific activity of the dose preparation were calculated based on the analytical data of radiochemical concentration of the dose preparation, the weight of [12C]NBPT used and the original specific activity of [14C]NBPT stated by the Sponsor. Radiochemical purity of the dose preparation was also determined prior to and after dosing lto examine the stability of the test materials in the dose preparation.
In both experiments, the specific density of the dose preparations (g/mL) was determined prior to and after dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not reported

- Concentration in vehicle:
Test material suspended in 0.75 % (w/v) methylcellulose (400 centipoise)/HPLC grade water.

- Amount of vehicle (if gavage):
Animals received the dose preparation or vehicle at the rate of approximately 5 mL/kg body weight.

STABILITY OF TEST MATERIALS IN DOSE PREPARATIONS
In the main experiment, radiochemical purities of the dose preparation were 94.9 and 93.9% prior to and after dosing, respectively. These results
indicated that the test material was stable in the dose preparation from preparation of the dose material until administration.

Duration and frequency of treatment / exposure:

A single oral dose of[14C]NBPT was administered for 168 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
Average of 250 mg/kg body weight (200 to 280 µCi/kg body weight).

No. of animals per sex per dose / concentration:

8 male SpragueDawley (Main study)

Control animals:

yes

Positive control reference chemical:

1 animal

Details on study design:

- Dose selection rationale:
The dose level, 250 mg [14C]-N-(n-butyl) thiophosphoric triamide/kg, has been reported (Hazelton Laboratories America, Inc. Document Number HLA 2591-100) to be a no-observable-effect level (NOEL) for cholinesterase depression.

- Rationale for animal assignment (if not random):
Animals were assigned to the treatment groups using a computer generated random permutation program. The animals were randomized to control bias in the study.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify):
Whole blood, spleen, kidneys, liver, mesenteric fat and carcass were analyzed for radiolabel content

- Time and frequency of sampling:
Blood samples were collected from each animal through the cannulae at 1,2,4,6,8, 10, 12, 16,24, 48, 72, 96 and 120 hours after dosing, and through the dorsal aorta at termination 168 hours after dosing, and then analyzed for radiolabel content. The peak concentration (Cmax) observed at any of the sampling times averaged 80.18 µg-equiv/g. The sampling time at which the peak concentration was observed (tmax), was 1 or 2 hours after dosing. After the tmax the blood level rapidly decreased to less than 14% of the Cmax by 24 hours after dosing and then declined to less than 5% by 168 hours.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify):
Whole blood, spleen, kidneys, liver, mesenteric fat and carcass were analyzed for radiolabel content

- Time and frequency of sampling:
Four of the eight rats had jugular cannulae surgically implanted prior to dosing. Blood samples were collected from each animal through the cannulaeat 1,2,4,6,8, 10, 12, 16, 24, 48, 72, 96 and l20 hours after dosing, and through the dorsal aorta at termination 168 hours after dosing, and then analyzed for radiolabel content.

- From how many animals: (samples pooled or not)
In the main experiment, test material was administered to eight rats, and urine, feces and cage rinses were collected for 168 hours. Four of the eight rats had jugular cannulae surgically implanted prior to dosing. Aliquots of blood were removed via the cannulae at 1,2,4,6,8, 10. 12, 16,24,48, 72, 96 and 120 hours following dosing. The samples of urine, feces, cage rinses and blood were analyzed for radiolabel content. The blood sample collection at 6 hours after dosing and the analysis of the samples were not indicated in the protocol, but the deviation did not affect the integrity of the study. Atennination 168 hours after dosing, the liver, mesenteric fat, kidney, spleen, whole blood and residual carcass were collected from all rats and then analyzed for radiolabel content. Metabolites in the urine were characterized and identified. Two control· rats, one for each experiment, received the vehicle. Samples collected from the control animals were used for the detennination of background radioactivity.

- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC)
High Performance Liquid Chromatography (HPLC)
Thin-Layer Chromatography (TLC)
Liquid scintillation counting (LSC)
High Pressure Liquid Chromatography/Mass Spectrometry (HPLC-MS)
Thin Layer Chromatography (TLC)
1H NUCLEAR MAGNETIC RESONANCE (1NMR)

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
Not reported

Statistics:

Not reported

Results and discussion

Preliminary studies:

In the pilot experiment, a single oral dose of [14C]-N-(n-butyl) thiophosphoric triamide [14C]NBPT) was administered at an average of 269 mg/kg body weight(263 µCi/kg body weight) to two male Sprague-Dawley rats. Expired air from each animal was passed through two C02 traps and one volatile organics trap. Anaverage of 40.22 percent of the administered dose (% AD) was excreted as 14COZ during,48 hours after dosing, while an average of 0.01% AD was excreted as volatile organics. Averages of 25.20 and 5.44% AD were excreted in urine and feces, respectively, during 48 hours after dosing. An average of 8.03% AD was r covered in cage rinses during 48 hours after dosing. The total excretion of radiolabel averaged 78.88% AD. The total recovery of radiolabel from each animal averaged 86.43% AD.
Therefore, the decision was made to use one additional 14C02 trap instead of a volatile-organics trap in the main experiment.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

There was a rapid absorption of [14C]NBPT equivalent residues into the systemic circulation following dosing (tmax: 1 or 2 hours).

Main ADME results 2

Type:

distribution

Results:

No other tissue was found to contain residues above 10 µg-equiv/g or 0.10% AD except carcass which contained 2.49% AD (5.83 µg-equiv/g).

Main ADME results 3

Type:

metabolism

Results:

Metabolism of [14C]NBPT leads to two metabolites which are readily excreted.

Main ADME results 4

Type:

excretion

Results:

The total excretion of radiolabel was an average of 80.12% AD during 168 hours after dosing. Both final metabolites of [14C]NBPT appear to be readily excreted in the urine.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

There was a rapid absorption of [14C]NBPT equivalent residues into the systemic circulation following dosing. After the peak blood level. the blood level indicated a biphasic declination. The first phase was rapid and the second was slower.

Details on distribution in tissues:

Only a selected number of tissues were analyzed for radiolabel content. Whole blood, spleen, kidney, liver, mesenteric fat and carcass were analyzed. The levels of radiolabel in whole blood, spleen, kidney, liver, mesenteric fat and carcass averaged 2.96, 5.05, 6.34, 12.64,9.88 and 5.83 /µg-equiv/g, respectively. The % AD in whole blood, spleen, kidney, liver, mesenteric fat and carcass averaged 0.10,0.01,0.03,0.29,0.01. and 2.49%, respectively.

Details on excretion:

Radiolabel in Expired Air:
Expired air from each animal was passed through three CO2 traps. An average of 35.42% AD was excreted in expired air as 14CO2 during 168 hours after
dosing. Most of the total 14CO2 excreted was found in expired air during the first 48 hours (an average of 34.50% AD).

Excretion of Radiolabel in Urine, Feces and Cage Rinses:
An average of 24.39% AD was excreted in urine during 168 hours after dosing. Most of the total radiolabel excreted was found in urine during the first 48 hours (an average of 23.88% AD).

An average of 8.65% AD was excreted in feces during 168 hours after dosing. Most of the total radiolabel excreted was found in feces during the first 48 hours (an average of 8.32% AD).

An average of 11.67% AD was recovered in the cage rinses during 168 hours after dosing. Most of the total radiolabel recovered was found in cage rinses during the first 48 hours after dosing (an average of 11.48% AD).

The total excretion of radiolabel was an average of 80.12% AD during 168 hours after dosing. Most of the total excretion was found in cage rinses during the first 48 hours after dosing (an average of 78.17% AD).

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#1

Toxicokinetic parameters:

Cmax: 80.18 µg-equiv/g.

Toxicokinetic parameters 2

Test no.:

#1

Toxicokinetic parameters:

Tmax: 1 or 2 hours after dosing

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Selected urine samples were analyzed by HPLC and the HPLC chromatograms showed that the urine contained two major components. HPLC analysis of the urine coinjected with standard [14C]NBPT showed that neither component was unmetabolized NBPT.
For convenience, the early eluting peak in the urine is designated as degradate 1 and the later eluting peak as degradate 2.

Degradate 1
HPLC retention time comparisons showed that degradate 1 from the rat urine and pH 3 hydrolysis product of NBPT eluted at similar retention times. The rat urine was fortified with pH 3 hydrolysis product and analyzed using HPLC system 2. Degradate 1 and pH 3 hydrolysis product coelute. In the hydrolysis
study3, the pH 3 hydrolysis product was identified as N-(n~butyl)thiophosphoricdiamide. A sample of degradate 1 was isolated from the rat urine and analyzed by LCIMS. The mass spectrum shows a peak with a protonated molecular ion M+1 at rn/z 169 for a molecular weight of 168. This is consistent with the molecular weight of the pH 3 hydrolysis product. (Other peaks in the spectrum can be attributed to matrix impurities). A further analysis using 1NMR was consistent with the presence of degradate 1.
The identification of degradate 1 as the hydrolysis product, N-(nbutyl) thiophosphoric diamide, was thus confirmed by mass spectral and NMR analysis.

Degradate 2
HPLC analysis of the rat urine showed degradate 2 to have a HPLC retention time of 16:06 minutes. Degradate 2 was isolated from the urine using HPLC system 3 (1 % acetic acid in water). Reanalysis of the isolated sample by HPLC showed degradate 2 to be extremely unstable at acidic pH <<4-5). Further analysis showed that isolated degradate 2 is hydrolysed in the presence of dilute acetic acid to give degradate 1 by HPLC. A sample of degradate 2 was isolated from rat urine in ion-pairing agents and water and analyzed by LCIMS. A scan from the mass spectrum shows a peak at mlz 344 which may represent the protonated molecular ion M+l for a molecular weight of 343. This could correspond to a glucuronic acid conjugate of NBPT. This glucuronic acid conjugate could form degradate I by hydrolysis as was seen in the HPLC analysis.
The glucuronic acid conjugate of NBPT is the proposed structure for degradate 2 based on these data.

Conclusion:
Two major metabolites of NBPT were found in the urine samples from rats which had been treated with [14C]NBPT. The metabolites were N-(n-butyl)-thiophosphoric diamide and the glucuronic acid conjugate of NBPT. NBPT was shown to degrade to N-(n-butyl)thiophosphoric diamide (degradate 1). Degradate 2 was tentatively identified as the glucuronic acid conjugate of NBPT which then hydrolyzes to N-(n-butyl)thiophosphoric diamide. Both metabolites appear to be readily excreted in the urine.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
[14C]NBPT was eliminated from male Sprague-Dawley rats during 168 hours after a single oral dosing, mainly via expired air as 14CO2 and urine, with averages of 35.42 and 24.39% AD, respectively. The total excretion of radiolabel averaged 80.12% AD during the 168-hour period after dosing. Rat liver contained the highest level of radiolabel of all measured tissues (12.64 µg-equiv/g), but still contained less than 0.29% AD. No other tissue was found to contain residues above 10 µg-equiv/g or 0.10% AD except carcass which contained 2.49% AD (5.83 f.1g-equiv/g). There was a rapid absorption of [14C]NBPT equivalent residues into the systemic circulation following dosing (tmax: 1 or 2 hours). After the peak blood level, the blood level indicated a biphasic declination. The first phase was rapid and the second was slower. Two major components were found in the urine of rats which had been treated with NBPT. NBPT was shown to degrade to N-(n-butyl)thiophosphoric diamide (degradate 1). Degradate 2 was tentatively identified as the glucuronic acid conjugate of NBPT which then hydrolyzes to N-(n butyl) thiophosphoric diamide. Both metabolites appear to be readily excreted in the urine.

Executive summary:

Introduction

The study was conducted according to the requirements of the the guideline EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics) of the United States of America. The study was designed to estimate the extent and rate of elimination of radiolabel in expired air, urine, feces and cage rinses from male Sprague-Dawley rats that received a singlp oral dose of [¹⁴C]-N-(n-butyl) thiophosphoric triamide. The liver, mesenteric fat, kidneys, spleen, whole blood and residual carcass also were analyzed for radiolabel content. The study also was designed to generate the blood pharmacokinetic data. In addition, metabolites of NBPT in the urine were characterized and identified.

Method

In the pilot experiment, a single oral dose of [¹⁴C]-N-(n-butyl) thiophosphoric triamide (¹⁴C]NBPT) was administered at an average of 269 mg/kg body weight (263 µCi/kg body weight) to two male Sprague-Dawley rats. Expired air from each animal was passed through two CO₂ traps and one volatile organics trap. An average of 40.22 percent of the administered dose (% AD) was excreted as ¹⁴CO₂ during, 48 hours after dosing, while an average of 0.01% AD was excreted as volatile organics. Averages of 25.20 and 5.44% AD were excreted in urine and feces, respectively, during 48 hours after dosing. An average of 8.03% AD was recovered in cage rinses during 48 hours after dosing. The total excretion of radiolabel averaged 78.88% AD. The total recovery of radiolabel from each animal averaged 86.43% AD.

In the main experiment, a single oral dose of [¹⁴C]NBPT was administered at an average of 252 mglkg body weight (206 µCi/kg body weight) to 8 male Sprague-Dawley rats. An average of 35.42% AD was excreted as ¹⁴CO₂ during 168 hours after dosing. Averages of 24.39 and 8.65% AD were excreted in urine and feces during 168 hours after dosing. An average of 11.67% AD was recovered in the cage rinses during 168 hours after dosing. The total excretion of radiolabel averaged 80.12% AD. Whole blood, spleen, kidneys, liver, mesenteric fat and carcass were analyzed for radiolabel content. Radiolabel in whole blood, spleen, kidneys, liver, mesenteric fat and carcass averaged 2.96,5.05,6.34, 12.64,9.88 and 5.83 µg-equiv/g, respectively. The %AD in whole blood, spleen, kidneys, liver, mesenteric fat and carcass averaged 0.10,0.01,0.03,0.29,0.01 and 2.49%, respectively. The total recovery of radiolabel averaged 83.04% AD.

Four of the eight rats had jugular cannulae surgically implanted prior to dosing. Blood samples were collected from each animal through the cannulae at 1,2,4,6,8, 10, 12, 16, 24, 48, 72, 96 and l20 hours after dosing, and through the dorsal aorta at termination 168 hours after dosing, and then analyzed for radiolabel content. The peak concentration (C_max) observed at any of the sampling times averaged 80.18 µg-equiv/g. The sampling time at which the peak concentration was observed (t_max), was 1 or 2 hours after dosing.

Results:

After the t_max the blood level rapidly decreased to less than 14% of the C_max by 24 hours after dosing and then declined to less than 5% by 168 hours.

Two major metabolites of NBPT were found in the urine samples from rats which had been treated with [¹⁴C]NBPT. The metabolites were N-(n-butyl)-thiophosphoric diamide and the glucuronic acid conjugate of NBPT

[¹⁴C]NBPT was eliminated from male Sprague-Dawley rats during 168 hours after a single oral dosing, mainly via expired air as ¹⁴CO₂ and via urine, with averages of 35.42 and 24.39% AD, respectively. The total excretion of radiolabel was an average of 80.12% AD during 168 hours after dosing. Rat liver contained the highest level of radiolabel of all measured tissues (12.64 µg-equiv/g), but still contained less than 0.29% AD. No other tissue was found to contain residues above 10 µg-equiv/g or 0.10% AD except carcass contained 2.49% AD (5.83 µg-equiv/g).

There was a rapid absorption of [¹⁴C]NBPT equivalent residues into the systemic circulation following dosing. After the peak blood level. the blood level indicated a biphasic declination. The first phase was rapid and the second was slower.

Conclusion

NBPT appears to be biotransformed to N-(n-butyl)thiophosphoric diamide and the glucuronic acid conjugate of NBPT which then hydrolyzes to N-(n-butyl)thiophosphoric diamide. Both metabolites appear to be readily excreted in the urine.