2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1 December 2003 to 27 February 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was conducted to assess and compare the absorption, distribution, metabolism, and elimination of [2,6-14C-ring labelled]test material and [2,6-14C-ring labelled]test material-triisopropanolamine (TIPA) following administration of single oral doses of those compounds. In this study, four male Fischer 344 rats were given a single oral dose of a solution that delivered a target dose of 50 mg 14C-test material/kg of body weight. A separate group of four male Fischer 344 rats were dosed with an equimolar amount of 14C-test material-TIPA at a target dose of 96 mg 14C-test material-TIPA/kg of body weight.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

EC Number:

604-721-7

Cas Number:

150114-71-9

Molecular formula:

C6H4Cl2N2O2

IUPAC Name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

Specific details on test material used for the study:

Purity: 94.5%

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 173 - 188 g
- Housing: Animals were housed one per cage in glass Roth-type metabolism cages. The metabolism cages were designed for the separation and collection of urine and faeces. Air was drawn through the metabolism cages at ca. 500 mL/minute
- Individual metabolism cages: yes
- Diet: ad libitum during the pre-exposure and study periods, except that at 16 hours prior to the scheduled oral administration of the dose solutions, all feed except for one pellet was withdrawn. Feed was returned to the animals at approximately 4-hour post-dosing.
- Water: municipal water, ad libitum
- Acclimation period: Animals were acclimated in metabolism cages for two days prior to dosing

CANNULATION
- Jugular Vein
The rats in which the blood plasma 14C-time-course was determined were obtained already cannulated in the jugular vein by the supplier.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 24 °C
- Humidity: 48 - 54 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The oral dose solutions were prepared as suspensions in 0.5 % aqueous methyl cellulose. Appropriate amounts of 14C-labelled and/or non-radiolabelled test material were added to deliver the target dose of 50 mg/kg body weight upon administration of ca. 5 g dose solution/kg body weight. To achieve that dose, the test material dose solution was prepared at a targeted concentration of 10.0 mg/g of dose solution (the actual analytically verified concentration was 9.9 mg/g). Similarly, appropriate amounts of 14C-labelled and/or non-radiolabelled test material-TIPA were added to deliver the target dose of 96 mg/kg body weight upon administration of ca. 5 g dose solution/kg body weight. To achieve that dose, the test material-TIPA dose solution was prepared at a targeted concentration of 19.2 mg/g of dose solution (the actual analytically verified concentration was 19.3 mg/g). Note that the 50 mg/kg dose level for test material, and the 96 mg/kg dose level for test material-TIPA were equimolar.
The amount of radioactivity administered was targeted at 125 μCi/kg for both test materials.
Due to the differences in the water solubilities of the two compounds, the physical properties of the two dose solution formulations were quite different. The triisopropanolamine salt of the test material is much more water soluble than the acid form of the test material and was completely dissolved in the aqueous-based dose solution. In contrast, much of the test material (acid) remained undissolved in the dose solution and that formulation would best be described as a suspension. Care was taken to uniformly re-suspend the test material in its dose formulation (shaking by hand) immediately prior to administering the gavage dose.

Duration and frequency of treatment / exposure:

Animals received a single oral dose of test material.

Doses / concentrations

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 males

Control animals:

no

Details on study design:

- Dose selection rationale: The dose level selected for this “bridging” pharmacokinetic/metabolism study was equivalent to the low dose that was orally administered in a previous study that was conducted to examine the absorption, distribution, metabolism, and elimination (ADME) of 14C-test material in the male Fischer 344 rat. In the present study, rats were dosed with equimolar amounts of either 14C-test material or 14C-test material-TIPA; specifically, at target doses of 50 mg/kg of the 14C-test material, and 96 mg/kg of 14C-test material-TIPA (ae conversion factor = 0.52).
- Rationale for animal assignment: Animals were selected based on patency of the jugular vein cannulae and randomly assigned to treatment groups using a computer-driven randomisation procedure.

Details on dosing and sampling:

- Blood Collection
The rats were fitted with indwelling jugular vein cannulae and plasma [14C]-concentration-time courses were constructed. Approximately 0.1 mL of blood was collected at the following times (0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, and 120 hours post-dosing) and plasma prepared by centrifugation. The plasma was analysed for radioactivity by liquid scintillation spectroscopy (LSS).

- Urine Collection
All urine voided during the study was collected in dry- ice cooled traps. The urine traps were changed at 6, 12, 24, 36 and 48 hours post-dosing followed by 24-hour intervals for the remainder of the study (the final collection time was at 120 hours post-dosing). The cages were rinsed with water at the time the traps were changed and the rinse collected. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analysed for radioactivity by LSS. Equal-volume aliquots of urine samples from the 0 - 6 hour and 6 - 12 hour collection intervals were pooled and stored at –80 °C and selected urine samples from those two collection intervals underwent chemical analysis.

- Faeces Collection
Faeces were collected in dry- ice chilled containers at 24-hour intervals throughout the duration of the study. An aqueous homogenate (~ 25 % w/w) was prepared, and weighed aliquots of these homogenates were combusted and quantitated for radioactivity by LSS. In addition, equal volume aliquots of faecal homogenates from each animal were taken from the 0 - 24 hour collection interval and pooled. These pooled samples were stored at –80 °C and designated for chemical analysis.

- Identification of Radiolabelled Compounds in Urine and Faeces
Following the chromatographic radioprofiling of urine and faecal extracts, representative samples were also analysed by liquid chromatography/mass spectrometry (LC/MS) in an attempt to confirm the identity of the major radiolabelled peak as parent compound(s) and to identify those metabolites that exceeded 5 % of the administered dose in the urine and faecal specimens.

- Terminal Sacrifice
At approximately 120- hour post-dosing, the animals were anesthetized with a CO₂/O₂ mixture and sacrificed by exsanguination. Following sacrifice the Roth cages were washed and the final cage wash analysed for radioactivity.
The following tissues were collected at sacrifice: gastrointestinal (GI) tract, plasma (terminal), residual carcass, kidney, skin, liver, spleen, perirenal fat, and whole blood (terminal).
The carcass, GI tract with contents, kidneys, liver, and whole blood were collected, homogenised (~ 33 % homogenate), and a weighed aliquot oxidised and analysed for radioactivity by LSS. A portion of the blood specimen was centrifuged to obtain plasma and the plasma analysed for radioactivity by LSS. The skin was removed from the carcass and a representative skin sample was directly oxidised and analysed for radioactivity by LSS. The remaining tissues (e.g., perirenal fat and spleen) were directly oxidised without homogenisation.

- Final Cage Wash
Following the terminal sacrifice of the animals, a final cage wash was performed. The final cage wash and contents were collected and the weight of the sample was determined. A weighed aliquot of the final cage wash was analysed for radioactivity.

- Plasma
Except for the terminal blood sample (of which a portion was used for plasma), blood samples were centrifuged to obtain plasma which was analysed for radioactivity to construct the plasma 14C-concentration-time courses.

- Control Samples
Control urine and faeces were collected in dry- ice chilled traps from one male rat not dosed with either of the 14C-labelled test materials. The control excreta was collected over a period of approximately 48 hours beginning on the day that the treatment animals were dosed at the time eartag numbers were assigned. The control animal(s) were sacrificed at study termination and blood collected by the same procedure as the dosed animals. No tissues were collected from the control animals.

Statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft® Excel® spreadsheets and databases in full precision mode (15 digits of accuracy). Pharmacokinetic parameters were determined, including AUC (area-under-the-curve) and elimination rate constants, using a spreadsheet-based (Microsoft® Excel®) pharmacokinetic computer modelling application (PK Solutions™, Summit Research Services, Ashland, Ohio). Additional statistical analyses were not performed (nor warranted) since a direct comparison of the 14C plasma concentration-time curves and the pharmacokinetic parameters derived from those curves together with comparisons of the rates and extent of 14C excretion in urine and faeces were sufficient to demonstrate the bioequivalence of the test material and the TIPA salt of the test material following oral administration of those compounds.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

test material was found to be readily absorbed

Main ADME results 2

Type:

excretion

Results:

test material was efficiently cleared through the urine and faeces

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The pooled faecal extracts and urine produced a single radiolabelled peak that eluted at a retention time equivalent to the corresponding standard for the parent material. These peaks represented 46.28 and 50.56 % of the administered doses in urine and faeces respectively.

Any other information on results incl. tables

Dose Administered

The actual test material concentrations (as mg test material/g of dose solution) and levels of radioactivity (as µCi of radioactivity/g of dose solution) of the prepared dose solution was within 1 and 19 % of the targeted test material concentration and radioactivity level, respectively. No signs of toxicity were observed following oral administration of the test material. The mean animal body weight was 0.178 kg; the average amounts of test material administered (per kg of body weight) was therefore within 5 % of the targeted dose level of 50 mg/kg. The average amount of radioactivity administered (per kg of body weight) was within 25 % of the targeted level of 125 µCi/kg bw. The slight difference between the targeted and actual administered doses of test material had no impact on the results of this study.

 

Distribution of Radioactivity in Excreta and Tissues

The overall recovery of radioactivity obtained from excreta, tissues, and final cage wash over the 120-hour collection period ranged from 93.0 to 101.5 % of the orally administered 14C-labelled test material (overall average recovery of 97.1 ± 3.5 %). Most of the recovered radioactivity was associated with excreta (average of 96.9 % of the administered 14C-activity), which represents 99.8 % of recovered radioactivity. Of the radioactivity recovered in excreta, slightly more 14C-activity was recovered in the faeces than in the urine. Faeces contained 43.8 to 53.6 % of the administered dose (average of 50.6 ± 4.6 %). The radioactivity recovered in urine (and associated cage rinses) ranged from 39.1 to 57.6 % of the administered dose (average of 46.3 ± 7.9 %). Except for a skin sample from one animal which accounted for 0.02 % of administered radioactivity, terminal blood from one animal which contained < 0.01% of administered dose and spleens from three animals which contained < 0.01 % of administered dose, none of the tissue samples that were collected at terminal sacrifice contained 14C levels exceeding the limit of quantitation (LOQ) from the group dosed with 14C-test material. In general LOQ’s were equivalent to radioactivity levels of≤0.01 % of administered dose. The radioactivity recovered in final cage washes accounted for 0.2 % of the administered dose.

 

Concentration-Time Course of Radioactivity in Plasma

The highest concentration of radioactivity was observed in the first plasma samples (taken at 0.25 hours post-dosing), with a concentration of 26 mg equivalent test material/g plasma (defined as the Observed Cmax values). The concentration of 14C activity in plasma decreased in a bi-exponential manner over the 48-hour time period in which samples were collected. Approximately a 10- fold decrease in plasma concentrations occurred during the first two hours post-dosing. By 48- and 24-hours post-dosing, plasma radioactivity levels were non-quantifiable for the 14C-test material group.

The plasma elimination half- lives estimated for the rapid initial (a), and slower terminal (b), phases of the curve were 0.3 and 8.8 hours for the 14C-test material dosed group.

The 12-hour area-under-the-plasma-curve (AUC) was calculated as 23.0 µg eq- hour/g plasma for the 14C-test material group. As stated previously, the highest concentration of 14C-activity in plasma was measured in the 0.25- hour samples. This suggests that absorption of 14C-test material following oral administration was very rapid, with the highest concentration of radioactivity in plasma (Cmax) occurring before the first plasma samples were taken. Because of the rapid absorption, there are essentially no concentration-time values defining the absorptive phase of the plasma time curve and the pharmacokinetic parameters which would define the absorptive phase (ka, actual Cmax, actual Tmax) cannot be calculated with a high degree of certainty. However, since plasma concentrations of radioactivity decreased with each successive time point beginning with the first set of samples taken at 0.25-hour, it is reasonable to state that the actual Tmax was less than 0.25 hours.

 

Urinary Excretion of Absorbed Radioactivity

The total amount of radioactivity recovered in urine over the entire 120- hour collection period accounted for 46 % of the administered dose of 14C-test material. Radioactivity was rapidly excreted following oral administration. The highest percentage of urinary radioactivity was excreted in the 0 to 6 hour collection interval, with 38 % of the administered dose of 14C-test material recovered in that initial interval (calculated from urine and rinses, combined). The radioactivity levels recovered in that initial 0 to 6 hour interval represent 83 % of the total urinary 14C-activity collected (over the entire 120 hour collection period). An additional 4 % of the administered dose was excreted in the 6 - 12 hour collection interval. By 24 hours post-dosing, 45 % of the dose was recovered in the urine. The radioactivity excreted in urine between 24 and 120 hours post-dosing represented only 2 % of the administered dose.

Urinary elimination half- lives estimated for the rapid initial (a), and slower terminal (b), phases of the curve were 2.8 and 7.8 hours for the 14C-test material-dose group.

 

Recovery of Radioactivity in Faeces

The total amount of radioactivity recovered in faeces over the entire 120 hour collection period accounted for 51 % of the administered dose of 14C-test material. The majority of the faecal radioactivity, was eliminated during the first 24-hour collection interval following dosing, with 49 % of the administered dose being recovered in that initial 0 to 24-hour interval. The radioactivity level recovered in the 0 to 24-hour interval represent 96 % of the total faecal 14C activity collected (over the entire 120-hour collection period).

 

Identification of Radiolabelled Peaks in Urine and Faecal Extracts

HPLC analyses with in-line radioactivity monitoring (RAM) of pooled urine (0 to 6 hour and 12 to 24 hour collection intervals) and faecal extracts from pooled 0 to 24 hour collection interval from the test material dosed group revealed a single radiolabelled peak that eluted at a retention time equivalent to an authentic standard of test material. Those single radiolabelled peaks represented 46.28 and 50.56 % of the administered doses in the urine and faecal samples, respectively.

Positive electrospray ionisation (PESI)-LC/MS/MS of an authentic standard of test material produced an essentially equivalent mass spectra that was representative of a spectrum expected for the test material. Analysis of pooled 6 to 12 hour urine samples (from rats dosed with test material by PESI-LC/MS/MS confirmed the identity of the major eluting peak (Peak A) as parent test material.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material was found to be readily absorbed and efficiently cleared through the urine and faeces. The overall potential for bioaccumulation is therefore low.

Executive summary:

This study was conducted to assess and compare the absorption, distribution, metabolism, and elimination of [2,6-14C-ring labelled]test material and [2,6-14C-ring labelled]test material-triisopropanolamine (TIPA) following administration of single oral doses of those compounds.

During the study, four male Fischer 344 rats were given a single oral dose of a solution that delivered a target dose of 50 mg 14C-test material/kg of body weight. A separate group of four male Fischer 344 rats were dosed with an equimolar amount of 14C-test material-TIPA at a target dose of 96 mg 14C-test material-TIPA/kg of body weight.

Both compounds were rapidly absorbed; the highest plasma concentrations of radioactivity (observed Cmax) for both compounds occurred in the first sample taken at 0.25 hours post-dosing and were 26 and 16 µg equivalent test material for the 14C-test material and 14C-test material-TIPA dose groups, respectively.

Pharmacokinetic parameters from the plasma time curves yielded the following values for 14C-test material and 14C-test material-TIPA, respectively: plasma AUC’s were 23.0 and 19.0 µg eq-hour/g of plasma; half-lives from the a phase of plasma elimination were 0.338 and 0.509 hours; and half-lives from the β phase of plasma elimination were 8.8 and 13.0 hours. The excretion of 38.3 % (for 14C-test material) and 34.6 % (for 14C-test material-TIPA) of administered radioactivity in urine within six hours of dosing confirms that the radiolabelled portion of the molecule was rapidly absorbed whether administered as test material acid or the TIPA salt of the test material. Based on the radioactivity recovered in urine through 120 hours, a minimum of 46.3 and 42.5 % of the orally administered 14C-test material and 14C-test material-TIPA was absorbed. The radioactivity associated with the two compounds was rapidly eliminated with 93.5 % (44.7 % in urine; 48.8 % in faeces), and 93.3 % (41.5 % in urine; 51.8 % in faeces) of the administered doses of 14C-test material and 14C-test material-TIPA, respectively, recovered in excreta within 24 hours post-dosing. Urinary elimination half-lives of the a phase were 2.8 hours and 2.5 hours for the 14C-test material and 14C-test material-TIPA dosed groups, respectively. Urinary elimination half-lives of the β phase were 7.8 hours and 10.7 hours for the 14C-test material and 14C-test material-TIPA dosed groups, respectively. The radiolabelled portion of both molecules was excreted essentially unchanged in urine and faeces. Except for a minor radioactive peak detected in the 0 to 6 hour pooled urine sample from the 14C-test material-TIPA dosed group (representing 0.34 % of administered dose), the only radiolabelled peak detected in analyses of urine and faecal extracts was confirmed as parent test material. The results from this study indicate that 14C-test material and 14C-test material-TIPA, when administered orally to rats, are bioequivalent in terms of absorption, distribution, metabolism, and excretion of the radiolabelled portion of the molecule(s).