Nitrapyrin

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 1987 to 11 August 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Objective of study:

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Males weighed 175 to 250 grams and females weighed 130 to 170 grams
- Housing: The rats given single doses were acclimated to glass Roth-type metabolism cages for 3 days prior to the oral administration of the radiotracer. Rats utilised for plasma 14C concentration determinations were fitted with an indwelling jugular vein cannula while under methoxyflurane anaesthesia and allowed approximately 2 days to recover from surgery. Those animals given the multiple doses (non-radiolabelled) were housed in wire mesh cages for the first 12 daily doses and then transferred to Roth cages for the last two daily doses of non-radiolabelled test material and the dose of 14C-nitrapyrin. Animals used for the determination of 14C concentrations in the plasma, liver, kidney and fat were maintained in wire mesh cages.
- Diet: Ad libitum, except that food was withdrawn for a 12 hour period prior to dosing with the radiotracer and 4 hours post-dosing.
- Water: Municipal drinking water ad libitum.
- Acclimation period: At least one week.

ENVIRONMENTAL CONDITIONS
- Temperature: Not specified; adequate environmental conditions concerning temperature for this species were maintained.
- Humidity (%): Not specified; adequate environmental conditions concerning relative humidity for this species were maintained.
- Photoperiod: 12 hour photocycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The 14C-test material dosing solutions were prepared in corn oil at target doses of 1 and 60 mg/kg with a volume of administration of 5 mL dosing solution/kg of body weight. The radiotracer was diluted with non-radiolabelled compound to obtain a target radioactivity level of approximately 5 uCi/mL of dosing solution for each of the two dose levels.

Duration and frequency of treatment / exposure:

1. Single oral administration of 14C-test material in corn oil by gavage (low, 1 mg/kg bw, and high, 60 mg/kg bw dose; 5 rats/sex/group).
2. Repeated 14 daily oral administrations of non-radiolabelled test material followed by a single oral dose of 14C-test material on day 15 (low dose; 5 rats/sex). These animals were used to study the absorption, tissue distribution, excretion and metabolism of the test material. All animals were terminated at 72 hrs after dosing.
3. Plasma concentrations were determined throughout 48 hr following single oral administrations of either the low or the high dose to 3 males per group fitted with indwelling jugular vein cannulae.
4. Three additional groups of male rats (3 animals/ time point) were administered the high dose and the concentrations of 14C in plasma, liver, kidney and fat were determined at 2, 10 and 24 hours post-dosing.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

The first group received a single oral administration of 14C-test material by gavage at a dose of approximately 60 mg/kg. An additional group fitted with indwelling jugular vein cannulae were also administered an oral gavage dose of 14C-test material at 60 mg/kg. Three additional groups of male rats (3 animals/ time interval) were administered 60 mg/kg of 14C-test material by gavage and the concentration of determined at 2, 10 and 24 hours post-dosing.

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

The second group was given 14C-test material as a single oral dose of approximately 1 mg/kg. The third group received 14 daily oral administrations of non-radiolabelled test material at the low dose level (1 mg/kg/day) followed by a single oral dose of 1 mg 14C-test material/kg on day 15. An additional group fitted with indwelling jugular vein cannulae were also administered an oral gavage dose of 14C-test material at 1 mg/kg.

No. of animals per sex per dose / concentration:

Groups 1 to 3 consisted of 5 animals per sex per dose.
Two additional groups of 3 male rats each were fitted with indwelling jugular vein cannulae and administered 14C-test material at approximately 1 and 60 mg/kg dose levels.
Three additional groups of male rats (3 animals/ time interval) were administered the high dose of 14C-test material (60 mg/kg).

Control animals:

no

Details on study design:

- Dose selection rationale: The low dose of 1 mg/kg was less than the no-observed-effect level in a 13 week dietary study, while the high dose of 60 mg/kg is above the dose (40 mg/kg/day) which elicited increases in relative and absolute kidney and liver weights.

Details on dosing and sampling:

All dosing solutions were analysed for test material and radioactivity by HPLC with UV detection and in a liquid scintillation counter, respectively. The dose solutions prepared for the 14C-plasma time-course experiments were 80 and 83 % of their target concentration for the 1 and 60 mg/kg dose groups, respectively. All other dose solutions were within 15 % of their target concentration. The difference between targeted and measured concentration in the dose solutions had no effect on the results obtained in this study. The test material was quite stable in the vehicle. After 16 days the concentration was within 3 % of the initial concentration found.

SAMPLE COLLECTION AND ANALYSIS
Rats were housed in glass Roth-type metabolism cages designed for the separate collection of urine, faeces, expired organics and 14CO2. All urine voided during successive 12 hour intervals was collected in dry ice chilled containers. Following collection of each urine specimen the cage was rinsed with distilled water. The weight of each 12-hour urine and cage rinse was determined, and weighed aliquots of each were mixed with Aqueous Counting Scintillant (ACS, Amersham Corp., Arlington Heights, IL) and analysed for radioactivity. In addition, approximately 500 µL of urine from each animal was taken from the 0-12 and 12-24 hour collections and pooled into their respective dose and sex groups. The pooled urine samples were then analysed by HPLC to determine metabolites. The identity of these metabolites was confirmed by GC/MS.
Faeces were collected at 24 hour intervals in dry ice chilled containers and stored frozen (-20 °C). An aqueous homogenate (33-50 % w/w) was prepared from each sample and weighed aliquots were oxidised in an OX-300 Biological Materials Oxidiser (R.J. Harvey Instrument Corporation, Hillsdale, NJ). The 14CO2 released upon oxidation was trapped in a solution of 1-methoxy-2-propanol:monoethanolamine (7:3) and combustion scintillant and analysed for radioactivity. Expired organics and 14CO2 were trapped in charcoal and a solution of 1-methoxy-2-propanol: monoethanolamine (7:3), respectively (60 mg/kg dose level only). The charcoal was desorbed overnight with toluene and an aliquot removed for 14C-analysis. The 14CO2 trapping solution was mixed directly with combustion scintillant and the radioactivity quantified.
Animals were anaesthetised with CO2 and exsanguinated 72 hours post-dosing. The following tissues were collected and analysed for radioactivity: Bone, brain, fat, gonads, heart, kidney, liver, lung, blood (plasma and RBC), skeletal muscle, spleen, skin and the remaining carcass. The fat, spleen (an aqueous homogenate was prepared for the 60 mg/kg group), heart, ovaries, skin, RBC, and bone were oxidised directly. Aqueous homogenates (33-50 % w/w) of all other tissues were prepared and aliquots of these homogenates were oxidised in a biological material oxidiser. The 14CO2 released on oxidation of these tissues was trapped and quantified as described for the faeces.
Blood samples, from cannulated male rats used for the 14C plasma time-course determinations were collected in heparinised capillary tubes. Samples were collected at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 18, 24, 30 and 48 hours after dosing from the indwelling jugular vein cannula and centrifuged to obtain the plasma. Weighed aliquots of plasma (approximately 0.06 g) were mixed directly with ACS and analysed for radioactivity.
Samples of liver, kidney, plasma and adipose tissue were taken at 2, 10 and 24 hours post-dosing from a separate group of male rats given a single high dose (60 mg/kg) of 14C-nitrapyrin. These tissues were analysed for 14C as previously described.

14C ANALYSIS
Radioactivity was quantified with a Mark III liquid scintillation counter (TM-Analytical, Elk Grove, IL). Count rates were corrected for background and quench (channel ratio technique) to convert counts per minute (cpm) to disintegrations per minute (DPM). Samples containing less than three times the average count rate of the concurrently run blanks were considered to contain insufficient radioactivity to reliably quantify. Using this criterion it was possible to quantify 14C in any tissue that contained ≥0.02 % dose/g wet weight.

HPLC ANALYSIS
The dose solution, radiotracer and the urine were analysed utilising a reverse-phase HPLC system. A NOVA-PAK C18, 4 µm particle size, 10 cm x 8 mm radial-pack column (Waters, Milford, MA) was used for the separations. An aliquot of each sample was first diluted in acetone (1:100 v:v) and 100 µL of the sample was injected onto the column. The mobile phase was maintained at 1 mL/min and programmed for a 30 minute gradient from 100 % solvent A (99:1 v:v mixture of water and acetic acid) to 100 % solvent B (94:5:1 v:v:v mixture of acetonitrile, water and acetic acid) using a linear ramp function. The UV absorbance and radioactivity in the column effluent were monitored with a Waters Lamda-Max Model 481 LC-Spectrophotometer set at 254 nm and a Packard Trace 7130 radioactivity monitor, respectively.

GC-MS ANALYSIS
The two fractions containing the 14C eluting from the HPLC column were collected, extracted in ethyl acetate and derivatised with ethereal diazomethane, and subjected to GC/MS analysis. Metabolite identification was accomplished by electron impact GC/MS on a Finnigan 46108. Separation was accomplished using a J&W 08-1301 capillary column (15 m x 0.32 mm with a 0.25 µm film). The chromatographic conditions were helium carrier at a 1.5 mL/minute flow rate; the capillary column temperature program was from 100 to 250 °C at 12.5 °C/minute.

Statistics:

KINETIC ANALYSIS
A two compartment pharmacokinetic model defined by the following equations was used to describe the time-course of radioactivity in the plasma of male rats given 14C-test material.
dA0/dt = -Dose x exp (-Ka x Time)
dA1/dt = Dose x Ka - A1 x (K12 + Ke) + A2 x K21
dA2/dt = A1 x K12 - A2 x K21
dAe/dt = A1 x Ke
Cp = A1/V1

A0 represents the amount of radioactivity in the gastrointestinal tract, while A1 and A2 define amounts of radioactivity in the central and peripheral compartments, respectively. The amount of test material excreted is represented by Ae. Cp represents the concentration of 14C in the plasma and the volume of distribution of the central compartment is defined by V1. The absorption (Ka), elimination (Ke) and transfer of 14C-material between the central and peripheral compartments (K12 and K21) are all described as first order processes. Optimised estimates for the model parameters (Ka, K12, K21 and Ke and V1) were obtained using SIMUSOLV; a computer program that contains a numerical integration, optimisation and graphical routine and that is available for license use from Mitchell and Gauthier Associates, Inc. Optimisation of the 1 mg/kg data set was done by varying all model parameters. The 60 mg/kg data set was optimised by varying the absorption rate constant (Ka) while maintaining all the other model parameters at the rates determined for the 1 mg/kg dose level.
The area under the plasma 14C-concentration time curve (AUC) was determined by the trapezoid rule. The plasma clearance and the half-lives for absorption and the rapid initial (α) and slow terminal (β) phases were calculated utilising the following relationships:

AUC (0 to ∞) = Σ(t2-t1)/2 x (CP1 + CP2) + CP48 hr/β

Plasma Clearance = Dose/AUC

α, β = 1/2 [(K12 + K21 + Ke) ± √[(K12 + K21 + Ke)^2 - (4 x K21 x Ke)]

t1/2 = ln 2/Ka

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption from the gut was rapid and complete (> 80 %), although slightly slower at the high dose level. No signs of toxicity were observed following oral administration of either the 1 or 60 mg/kg dose levels.

Details on distribution in tissues:

The tissues contained only a minimum amount (<1 %) of radioactivity at 72 h.
The liver accounted for most of the radioactivity averaging 0.66 % of the dose. Small but quantifiable amounts of radioactivity were also found in the kidneys, lung, plasma and RBCs, and collectively these tissues accounted for an average of 0.11 % of the dose.
No differences in the overall distribution of the radioactivity in the excreta and tissues at 72 hours post-dosing were noted between the single and multiple exposures, between the low and high dose level, or genders.
At 2 and 10 hours after a single oral high dose to male rats, the highest concentration was found in the fat. The plasma and kidney contained approximately equal concentrations and the liver was approximately 1/3 the plasma 14C-concentration. At 24 and 72 hours, the highest concentration was found in the liver, although by 72 hours the concentration of 14C activity in all tissues was minimal.

Details on excretion:

The urine was the principal route of elimination and accounted for over 79 % of the administered dose (range 79.6 to 85.5 %) with cage wash representing an additional average of 0.44 % of the administered radioactivity (range 0.10 to 0.67 %), while excretion in faeces accounted for between 11.0 to 14.2 %. Independently of dosing regimen (single or repeated), dose level or genders, most radioactivity was excreted within the first 24 h. Quantitative recovery was obtained within 72 h (range 94.81 to 99.24 %).

Toxicokinetic parameters

Toxicokinetic parameters 1

Toxicokinetic parameters:

other: half life of 1.22 h for the 1 mg/kg dose group

Toxicokinetic parameters 2

Toxicokinetic parameters:

other: half life of 3.19 h for the 60 mg/kg dose group

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The urine from all treatment groups contained only two metabolites, identified as 6-chloropicolinic acid (6-CPA) and the glycine conjugate of 6-CPA (6-CPA-Gly). No unchanged test material was found in the urine. The structure of these excretion products was confirmed by mass spectrometry of their methyl ester derivatives. All of the test material excreted via the urine was metabolised to 6-CPA and approximately half of the 6-CPA was then conjugated with glycine.

Any other information on results incl. tables

Discussion

The time-course of 14C-test material in the plasma of the male rat was well described by a 2-compartment pharmacokinetic model. The optimised pharmacokinetic model parameters fit both the 1 and 60 mg/kg dose plasma time-course. However, the absorption rate was approximately 2.6 fold slower in the 60 mg/kg dose group, based on the ratio of the absorption rate constants. Although the absorption rates varied between doses the AUC·s were proportional to the dose indicating that the amount of test material absorbed was comparable at both doses.

The plasma clearance of 14C at both dose levels was approximately 2.70 g min^-1 kg^-1. The glomerular filtration rate in the rat is 3.4 g min^-1 kg^-1. Thus, the renal clearance of radioactivity could be explained by glomerular filtration.

The plasma 14C time-course data demonstrated a slower rate of absorption at the 60 mg/kg dose. The passage of a xenobiotic from the gastrointestinal tract into the blood stream is primarily by passive diffusion. Chemicals and drugs exist in different ionisation states based on their particular pKa. This is of crucial importance since only the neutral (non-ionised) form of the compound is readily absorbed by diffusion across a lipid membrane. The acidity of the digestive tract gradually decreases in moving from the stomach toward the large intestines. Therefore, the extent of absorption of any chemical is a function of intestinal transit time, pKa and the pH of the intestines. The different rates of absorption for the 1 and 60 mg/kg dose groups may be due to differences in pH-partitioning through the various intestinal compartments. Additionally, alterations in gastric emptying and transit time, uncertain chemical stability in an acidic environment or the binding with other materials in the gastrointestinal tract may play contributing roles altering the absorption rate.

After the test material was absorbed it was rapidly metabolised and excreted. At 72 hours post-dosing, over 94 % of the administered dose was recovered at both doses and the principle excretory route was via the urine. The faeces represented a minor excretory pathway, but together with the urinary recovery accounted for approximately 99 % of the recovered dose. Repeated administrations did not alter the metabolism or excretion rates in comparison with the single 1 and 60 mg/kg doses. These data indicate that there are no significant differences in the overall disposition of the test material between dose levels and/or sexes.

The test material is lipophilic in character and could explain why the fat contained the highest 14C concentration at 2 and 10 hours post-dosing. However, the extensive analysis of all major organ systems for radioactivity at 72 hours post-dosing indicated that less than 1 % of the administered dose was recovered in the liver, kidney, lung and blood. These data suggest that the test material is unlikely to accumulate even in the fat of the rat.

The urinary 14C excretion time-course demonstrated a slower excretion rate for the 60 mg/kg dose group. This slower initial excretion rate was primarily attributable to the slower rate of absorption and not due to the saturation of metabolism or the urinary excretion pathways. This conclusion is consistent with the plasma 14C time-course data and is supported by the proportional AUCs and plasma clearance data.

The two urinary metabolites identified were 6-CPA and the glycine conjugate of 6-CPA (6-CPA-Gly) which accounted for 100 % of the urine radioactivity. The extent of glycine conjugation in this study ranged from 31 to 82 % of the urinary 14C activity. These data suggest that the test material and/or its metabolites are rapidly excreted and are thus likely to have a low potential to accumulate upon repeated administration.

In summary, these data indicate that the test material is readily absorbed after oral administration (>80 %). The 14C plasma-time course data exhibited a biexponential decrease in radioactivity and was adequately described by a 2-compartment pharmacokinetic model. The primary difference between the 1 and 60 mg/kg dose pharmacokinetics was the slower rate of absorption for the high dose groups (2.6 fold). However, after being absorbed both dose levels were rapidly eliminated and over 94 % of the administered dose was excreted within 72 hours post-dosing. The principal excretory route was via the urine (>80 %) with the faeces representing a minor excretory pathway (>11 %). In addition, the disposition of 14C-tets material was unaffected by prior exposure to 1 mg/kg/day (14 days) of the test material. Two urinary metabolites, 6-CPA and 6-CPA-Gly were identified and accounted for 100 % of the radioactivity in the urine. The results of these experiments indicate that the test material is rapidly metabolised and excreted as 6-CPA and 6-CPA-Gly.

Table 1: Distribution of Radioactivity Recovered 72 Hours after Oral Administration

	Percent of Administered Dose
	1 mg/kg		60 mg/kg		1 mg/kg (Multiple)
	Male	Female	Male	Female	Male	Female
Urine	82.73 ± 1.52	79.56 ± 5.34	82.64 ± 3.53	83.91 ± 2.25	85.48 ± 1.21	84.91 ± 0.99
Faeces	11.84 ± 2.01	13.63 ± 2.18	13.06 ± 1.15	14.16 ± 1.97	11.04 ± 0.99	11.51 ± 1.59
Tissue	0.93	0.95	0.55	0.51	0.85	0.79
Cage Wash	0.36 ± 0.14	0.67 ± 0.46	0.48 ± 0.21	0.66 ± 0.40	0.10 ± 0.09	0.36 ± 0.46
TOTAL	95.86	94.81	96.73	99.24	97.47	97.57

Values represent mean ± S.D. of 5 animals

 

Table 2: Amount of Radioactivity in the Tissues 72 Hours after Oral Administration

	Percent of Administered Dose
	1 mg/kg		60 mg/kg		1 mg/kg (Multiple)
	Male	Female	Male	Female	Male	Female
Kidney	NQ	0.058 ± 0.004	0.050 ± 0.004	NQ	0.042 ± 0.003	0.058 ± 0.009
Liver	0.838 ± 0.46	0.781 ± 0.089	0.499 ± 0.044	0.484 ± 0.047	0.735 ± 0.026	0.643 ± 0.061
Lung	0.070 ± 0.25	0.072 ± 0.015	NQ	NQ	0.043 ± 0.003	0.054 ± 0.005
Plasma	0.002 ± 0.001	0.006 ± 0.001	NQ	0.006 ± 0.001	0.004 ± 0.001	0.005 ± 0.002
RBC	0.021 ± 0.005	0.028 ± 0.003	NQ	0.018 ± 0.004	0.023 ± 0.011	0.025 ± 0.003
TOTAL	0.931	0.945	0.549	0.508	0.847	0.785

Values represent mean ± S.D. of 5 animals

NQ = Non-quantifiable. Samples contained less than 3 times background radioactivity were considered non-quantifiable. The amount of radioactivity in the following tissues was NQ for all animals: Bone, brain, carcass, fat, gonads, heart, muscle, skin and spleen.

 

Table 3: Observed and Model Prediction Concentrations of Radioactivity in the Plasma Following Oral Administration to Male Rats at Target Doses of 1 and 60 mg/kg

Collection Time (hours post-dosing)	µg Equivalents Test Material/g Plasma
	1 mg/kg		60 mg/kg
	Observed	Predicted	Observed	Predicted
0.25	0.212 ± 0.015	0.176	8.108 ± 0.260	4.376
0.50	0.320 ± 0.055	0.315	13.548 ± 0.864	8.194
0.75	0.425 ± 0.082	0.424	16.252 ± 3.595	11.51
1.0	0.453 ± 0.070	0.507	18.799 ± 3.078	14.37
2.0	0.599 ± 0.142	0.660	23.849 ± 2.093	22.11
4.0	0.525 ± 0.143	0.569	20.308 ± 2.943	26.29
6.0	0.409 ± 0.042	0.378	15.532 ± 1.739	23.59
8.0	0.264 ± 0.133	0.231	14.142 ± 1.753	18.95
12.0	0.075 ± 0.017	0.082	14.570 ± 1.246	10.59
18.0	0.024 ± 0.008	0.023	5.542 ± 1.046	3.983
24.0	0.010 ± 0.002	0.011	2.126 ± 0.705	1.605
30.0	0.008 ± 0.002	0.007	0.798 ± 0.039	0.7816
48.0	0.003 ± 0.001	0.003	0.225 ± 0.033	0.2271

Observed values represents mean ± S.D. of 3 animals per group

Predicted concentration based on model parameters in Table 4

 

Table 4: Parameters for 2-Compartment Model that Best Described the Time Course in the Male Rat

Parameters	1 mg/kg	60 mg/kg
Absorption (Ka, hr^-1)	0.568	0.217
Transfer from 1st to 2nd Compt (K12, hr^-1)	0.0264	0.0264
Transfer from 2nd to 1st Compt (K21, hr^-1)	0.0529	0.0529
Elimination (Ke, hr^-1)	0.285	0.285
Volume of Central Compt (V1, mL/kg)	579	579
Half-life of Absorption Phase (t1/2, hr)	1.22	3.19
Half-life of Initial Phase ((t1/2)α, hr)	2.19	2.19
Half-life of Terminal Phase ((t1/2)β, hr)	14.56	14.56
Area Under Plasma Curve (AUC, µg g^-1 hr)	4.96	307.51
Plasma Clearance (g hr^-1 kg-^1)	161.29	162.60

The 1 mg/kg data set was optimised by allowing all model parameters (Ka, K12, K21, Ke and V1, to vary. The 60 mg/kg dose group was optimised by varying only the absorption rate (Ka) while holding all other parameters at the values determined for the 1 mg/kg dose.

 

Table 5: Concentration of Radioactivity in the Plasma, Liver, Kidney and Fat at 2, 10, 24 and 72 Hours After Oral Administration to Male Rats at a Dose of 60 mg/kg

Collection Time (hours post-dosing)	µg Equivalents of Test Material/g Tissue
	Plasma	Liver	Kidney	Fat
2	38.55 ± 2.11	11.60 ± 1.06	26.49 ± 1.99	72.46 ± 4.04
10	31.42 ± 3.80	8.84 ± 3.18	35.47 ± 1.01	183.10 ± 11.72
24	3.76 ± 0.45	6.83 ± 3.18	4.13 ± 0.49	4.22 ± 0.95
72	NQ	2.02 ± 0.13	1.00 ± 0.08	NQ

Values represent mean ± S.D. of 3 animals/time interval (2, 10 and 24 hours). The 72 hour data are from the balance study and are for 5 animals.

NQ = Non-quantifiable. Samples containing less than 3 times background radioactivity were considered non-quantifiable.

 

Table 6: Excretion of Radioactivity in the Urine and Cage Rinse after Oral Administration in Male and Female Rats

Collection interval (hours)	Percent of Administered Dose
	1 mg/kg		60 mg/kg		1 mg/kg (Multiple)
	Male	Female	Male	Female	Male	Female
0-12	69.59 ± 2.70	63.93 ± 4.70	41.60 ± 7.43	38.57 ± 14.79	73.61 ± 2.98	71.70 ± 2.95
12-24	10.88 ± 1.84	12.74 ± 2.44	36.36 ± 8.56	38.74 ± 12.65	9.68 ± 2.40	10.45 ± 2.67
24-36	1.23 ± 0.16	1.51 ± 0.56	3.41 ± 0.66	4.77 ± 2.90	1.21 ± 0.22	1.55 ± 0.48
36-48	0.57 ± 0.13	0.77 ± 0.46	0.80 ± 0.26	1.19 ± 0.27	0.51 ± 0.09	0.63 ± 0.16
48-60	0.26 ± 0.05	0.37 ± 0.10	0.30 ± 0.03	0.33 ± 0.08	0.28 ± 0.03	0.35 ± 0.12
60-72	0.21 ± 0.07	0.23 ± 0.07	0.18 ± 0.04	0.32 ± 0.18	0.19 ± 0.04	0.24 ± 0.04
TOTAL	82.73 ± 1.52	79.56 ± 5.34	82.64 ± 3.53	83.91 ± 2.25	85.48 ± 0.21	84.91 ± 0.99

Value represents mean ± S.D. of 5 animals

 

Table 7: Excretion of Radioactivity in the Faeces after Oral Administration to Male and Female Rats

Collection interval (hours)	Percent of Administered Dose
	1 mg/kg		60 mg/kg		1 mg/kg (Multiple)
	Male	Female	Male	Female	Male	Female
0-24	10.13 ± 1.90	11.12 ± 1.31	10.04 ± 1.30	10.98 ± 2.92	9.37 ± 1.21	8.44 ± 2.34
24-48	1.39 ± 0.27	2.13 ± 1.31	2.58 ± 0.60	2.60 ± 1.21	1.51 ± 0.32	2.75 ± 1.42
48-72	0.32 ± 0.14	0.38 ± 0.19	0.44 ± 0.23	0.57 ± 0.22	0.16 ± 0.02	0.33 ± 0.16
TOTAL	11.84 ± 2.01	13.63 ± 2.18	13.06 ± 1.15	14.16 ± 1.97	11.04 ± 0.99	11.51 ± 1.59

Value represents mean ± S.D. of 5 animals

 

Table 8: The Percent of 14C in the Urine as 6-CPA or 6-CPA-Gly at 0-12 and 12-24 Hours Post-Dosing in Male and Female Rats

Metabolite	Percent of 14C in Urine
	1 mg/kg		60 mg/kg		1 mg/kg (Multiple)
	Male	Female	Male	Female	Male	Female
	0-12 Hour urine collection
6-CPA	40.3	27.7	45.2	40.9	29.5	18.3
6-CPA-Gly	59.7	72.3	54.8	59.1	70.5	81.7
	12-24 Hour urine collection
6-CPA	52.5	46.5	69.0	54.0	41.6	20.5
6-CPA-Gly	47.5	53.5	31.0	46.0	58.4	79.9

Aliquots of urine were pooled from 5 animals from each group and analysed by HPLC.

Only two metabolites were found and were identified as 6-chloropicolinic acid (6-CPA) and the glycine conjugate of 6-CPA (6-CPA-Gly).

Applicant's summary and conclusion

Conclusions:

These data indicate that the test material is readily absorbed after oral administration (>80 %). The principal excretory route was via the urine (>80 %) with the faeces representing a minor excretory pathway (>11 %).

Executive summary:

This study was conducted to provide data on the pharmacokinetics of the test material in accordance with the standardised guideline EPA OPP 85-1 under GLP conditions. 14C-test material was administered orally to groups of 5 male and 5 female rats as a single dose at approximately 1 and 60 mg/kg body weight and as a multiple 1 mg/kg non-radiolabelled dose for 14 days followed by a single oral dose of 1 mg 14C-test material/kg on day 15. Two additional groups of male rats were used to obtain 14C-plasma time-course and 14C-tissue distribution data.

The results indicate that although both doses were readily absorbed (>80 %), the low dose was absorbed at a faster rate than the high dose (t1/2 = 1.2 and 3.2 hours, respectively). The radioactivity was cleared from the plasma in a biexponential manner; half-lives for the rapid (initial) and slow (terminal) phases were 2.2 and 14.6 hours, respectively. Over 94 % of the dose was excreted in 72 hours.

The principle route of excretion was the urine (>80 %), which together with the faecal excretion (>11 %) accounted for approximately 99 % of the recovered dose. The tissues contained only a minor (<1 %) amount of 14C activity at 72 hours. HPLC and GC/MS analysis of urine samples identified two urinary metabolites as 6-chloropicolinic acid (6-CPA) and the glycine conjugate of 6-CPA (6-CPA-Gly), which accounted for 100 % of the urinary radioactivity. These data demonstrate that the test material was well absorbed from the GI tract. However, the 60 mg/kg dose was absorbed at a slower rate than the 1 mg/kg dose. After being absorbed it was rapidly metabolised and excreted principally in the urine as 6-CPA and 6-CPA-Gly. The elimination and final disposition of 14C-test material was unaffected by either dose level or prior exposure.