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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Qualifier:
according to
Guideline:
other: FIFRA Guideline No. 85-1
Qualifier:
according to
Guideline:
other: EEC Directive 87/302/EEC Toxicokinetics
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Triisopropanolamine salt of 2,4-Dichlorophenoxyacetic acid (2,4-D TIPA salt)

A liquid sample of 2,4-D-TIPA salt was obtained from DowElanco, Midland, MI. Assay of this sample by nuclear magnetic resonance (NMR) indicated the following composition (w/Ww%): 22.0 +/- 0.9% H20, 40.3 +/- 1.5% 2,4-D and 37.1 +/- 1.5% TIPA. The molar ratio of amine to acid was 1.06 +/- 0.5. The test material was also characterized by high performance liquid chromatography (HPLC) and found to be 38.7 +/- 0.1% 2,4-D (acid equivalents) or 72.2 +/-0.2% 2,4-D TIPA salt at the 95% confidence level. Reanalysis of the sample indicated no significant change in the purity.

Triisopropanolamine-1-14C (14C-TIPA) with a specific activity of 31.9mCi/mmol, was obtained from DowElanco, Midland, MI. The 14C-TIPA was diluted with hexane/benzene (1:1; v/v) prior to being analyzed for radiochemical purity. The radiochemical purity of 14C-TIPA was determined to be 97.5 +/- 1.1%.
Radiolabelling:
yes
Remarks:
14C labeled

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Male Fischer 344 rats (10 weeks old) were purchased from the Charles River Breeding Laboratories (Kingston, NY) and weighed between 170 and 183 grams when dosed. Care and husbandry of these animals was in accordance with the Standard Operating Procedures of the Laboratory (Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC)). Upon arrival at the laboratory, the rats were examined by a veterinarian and found to be in good general health. The rats were then acclimated to the laboratory for at least one week prior to use. The rooms in which the animals were housed had a 12-hr photocycle, and were designed to maintain adequate temperature and relative humidity for rats. Prior to the start of the experiment, rats were selected from those available using a computer driven randomization procedure and were individually identified by numbered metal ear tags. The animals were acclimated overnight to glass Roth-type metabolism cages. On the next day, they were anesthetized with methoxyflurane and an indwelling jugular vein cannula implanted (Harms and Ojeda, 1974). The rats were then allowed approximately 1 day to recover from surgery prior to administration of the oral dose. Certified rodent chow #5002 (Purina Mills Inc., St. Louis, MO) and municipal drinking water were provided ad libitum, except that food was withdrawn from these animals approximately 17 hr prior to dosing and returned approximately 4 hr post-dosing. Feed and water were analyzed in accordance with the Standard Operating Procedures of The Toxicology Research Laboratory.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Rats were housed in Roth-type metabolism cages designed for the separate collection of urine, feces, 14CO2 and expired organic 14C. Air was drawn through the cages at approximately 500 ml/min. The air, upon exiting the cage, was passed through a trap containing about 10 grams of charcoal to capture expired organic 14C and then through a trap containing about 150 ml of 3:7 (V/V) monoethanolamine: 1-methoxy-2-propanol to capture expired 14CO2. The charcoal trap was changed at 12 hours post-dosing and analyzed for radioactivity. There was insufficient radioactivity in the 0-12 hr charcoal trap to quantify (i.e., < twice background), thus, collection of the charcoal traps was discontinued. The 14CO2 traps were changed at 12, 24, 36, 48 and 72 hours post-dosing. Following collection, the weight of the 14CO2 trap solution was determined, and a weighed aliquot of the trap solution was mixed with liquid scintillation fluid (12% SPECTRAFLUOR: 22% 1-methoxy-2-propanol: 66% toluene) and analyzed for radioactivity.

Blood samples of about 0.2 ml each were drawn from the jugular cannula at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 18, 24, 48 and 72 hr post-dosing using a syringe. Following collection of each blood sample, samples were centrifuged to obtain plasma. The plasma was subsequently weighed, mixed with Aquasol liquid scintillation fluid and analyzed for radioactivity.

Urine was collected in dry-ice chilled containers that were changed at 6, 12, 24, 48 and 72 hr post-dosing. To minimize carry over between specimens, the cage was rinsed with distilled water following collection of each urine specimen. The weight of each urine and cage rinse specimen was determined, and weighed aliquots of each specimen were mixed with Aquasol liquid scintillation fluid (NEN Research Products, Boston, MA) and analyzed for radioactivity. The urine and cage rinse radioactivity was combined for each collection interval and expressed as radioactivity excreted in the urine. In addition, pooled urine samples were prepared from the 0-6 and 6-12 hr collection intervals by mixing the 0.5 ml of urine from each rat. These pooled urine samples were stored frozen (-80C) until analyzed for 2,4-D and for 14C-TIPA and metabolites.

Feces were collected at 24-hr intervals in dry-ice chilled containers. The feces were weighed and an aqueous homogenate (~33% w/w) prepared. Weighed aliquots (~95 mg) of these homogenates were placed in scintillation vials, mixed with 1.5 ml Soluene-350 (Packard Instruments, Downers Grove, IL) and incubated at 40C for 1-2 hr. Then, the sample was decolorized following the addition of 0.75 ml isopropanol and 0.3 ml of 30% hydrogen peroxide, and incubated at 40C for an additional 1-2 hr. Finally, the sample was mixed with 15 ml Hionic-Fluor liquid scintillation fluid (Packard Instruments, Downers Grove, IL). Glacial acetic acid was added to 100 ul increments to minimize photo-chemiluminescence.

Animals were euthanatized with CO2 and exsanguinated 72 hr post-dosing. The following tissues were collected and analyzed for radioactivity: liver, kidneys, perirenal fat, skin and remaining carcass. Weighed aliquots of skin and aqueous homogenates (~33% w/w) of the carcass and other tissues were solubilized and radioactivity quantified in a manner similar to that described for the feces except the samples did not require decolorization.
Duration and frequency of treatment / exposure:
Single oral gavage administration
Doses / concentrations
Dose / conc.:
10.7 mg/kg bw/day
Remarks:
2,4-D-TIPA (0.045 mmol/kg bw), equivalent to 10 mg TIPA/kg bw
No. of animals per sex per dose:
4 males
Control animals:
no
Positive control:
no
Details on study design:
Rats were housed in Roth-type metabolism cages designed for the separate collection of urine, feces, 14CO2 and expired organic 14C. Air was drawn through the cages at approximately 500 ml/min. The air, upon exiting the cage, was passed through a trap containing about 10 grams of charcoal to capture expired organic 14C and then through a trap containing about 150 ml of 3:7 (V/V) monoethanolamine: 1-methoxy-2-propanol to capture expired 14CO2. The charcoal trap was changed at 12 hours post-dosing and analyzed for radioactivity. There was insufficient radioactivity in the 0-12 hr charcoal trap to quantify (i.e., < twice background), thus, collection of the charcoal traps was discontinued. The 14CO2 traps were changed at 12, 24, 36, 48 and 72 hours post-dosing. Following collection, the weight of the 14CO2 trap solution was determined, and a weighed aliquot of the trap solution was mixed with liquid scintillation fluid (12% SPECTRAFLUOR: 22% 1-methoxy-2-propanol: 66% toluene) and analyzed for radioactivity.

Blood samples of about 0.2 ml each were drawn from the jugular cannula at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 18, 24, 48 and 72 hr post-dosing using a syringe. Following collection of each blood sample, samples were centrifuged to obtain plasma. The plasma was subsequently weighed, mixed with Aquasol liquid scintillation fluid and analyzed for radioactivity.

Urine was collected in dry-ice chilled containers that were changed at 6, 12, 24, 48 and 72 hr post-dosing. To minimize carry over between specimens, the cage was rinsed with distilled water following collection of each urine specimen. The weight of each urine and cage rinse specimen was determined, and weighed aliquots of each specimen were mixed with Aquasol liquid scintillation fluid (NEN Research Products, Boston, MA) and analyzed for radioactivity. The urine and cage rinse radioactivity was combined for each collection interval and expressed as radioactivity excreted in the urine. In addition, pooled urine samples were prepared from the 0-6 and 6-12 hr collection intervals by mixing the 0.5 ml of urine from each rat. These pooled urine samples were stored frozen (-80C) until analyzed for 2,4-D and for 14C-TIPA and metabolites.

Feces were collected at 24-hr intervals in dry-ice chilled containers. The feces were weighed and an aqueous homogenate (~33% w/w) prepared. Weighed aliquots (~95 mg) of these homogenates were placed in scintillation vials, mixed with 1.5 ml Soluene-350 (Packard Instruments, Downers Grove, IL) and incubated at 40C for 1-2 hr. Then, the sample was decolorized following the addition of 0.75 ml isopropanol and 0.3 ml of 30% hydrogen peroxide, and incubated at 40C for an additional 1-2 hr. Finally, the sample was mixed with 15 ml Hionic-Fluor liquid scintillation fluid (Packard Instruments, Downers Grove, IL). Glacial acetic acid was added to 100 ul increments to minimize photo-chemiluminescence.

Animals were euthanatized with CO2 and exsanguinated 72 hr post-dosing. The following tissues were collected and analyzed for radioactivity: liver, kidneys, perirenal fat, skin and remaining carcass. Weighed aliquots of skin and aqueous homogenates (~33% w/w) of the carcass and other tissues were solubilized and radioactivity quantified in a manner similar to that described for the feces except the samples did not require decolorization.

14C Analysis: Radioactivity was quantified with a Beckman LS 3801 liquid scintillation counter (Beckman Instruments, Fullerton, CA). Counts per minute were corrected for background and quench, and converted to disintegrations per minute (dpm). At least one sealed standard was counted with each group of samples to monitor the performance of the liquid scintillation counter.

Mass Spectrometry Analysis of Urinary 14C-TIPA: Aliquots of the 0-6 and 6-12 hr pooled urine samples were made basic with 5N NaOH and extracted with methylene chloride. The extracts were then blown to dryness with nitrogen, derivatized with TFAI and analyzed using GC/MS.

Gas Chromatography of Urinary Metabolites of 14C-TIPA: The TFAI derivatized methylene chloride extracts were also analyzed by GC with a Radiomatic FLO-ONE/BETA Model GCR radioactivity-GC-detector.
Details on dosing and sampling:
The dose solution was prepared by adding a measured volume of 14C-TIPA to a 5 ml volumetric flask and removing the hexane/benzene solvent by evaporation. Then, a measured volume of the non-radiolabeled 2,4-D-TIPA was added and the volumetric flask was diluted to volume with distilled water. Targeted concentrations of TIPA and radioactivity in this solution were 5.35 mg TIPA and 60 uCi of 14C per ml. Administration of this dose solution at the rate of 2 ml/kg body weight resulted in a targeted dose of 10.7 mg TIPA and 20-30uCi of 14C per animal. Radioactivity in the dose solution was quantified using a liquid scintillation counter. The concentration of TIPA in the dose solution was calculated to be 5.55 mg/ml based on the amount of 2,4-D (5.18 mg TIPA/ml; molar ratio of 2,4-D to non-radiolabeled TIPA was 0.92) and 14C-TIPA (0.37 mg/ml) in the dose solution.

The rats were weighed and based on their body weight a measured volume of the dose solution was administered by gavage using a glass syringe and stainless steel feeding needle (Popper & Sons, Inc., New Hyde Park, NY). The quantity of dose solution actually administed was determined by weighing the syringe prior to and following dosing.
Statistics:
Descriptive statistics were calculated (mean +/- S.D.) where appropriate. The plasma 14C-concentration time data were described by a polyexponential equation using the method of residuals (Gibaldi and Perrier, 1975).

Results and discussion

Preliminary studies:
No preliminary studies were conducted since information was available from previous studies

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The highest concentration of radioactivity (4.48 +/- 1.19 ug eq/g plasma) was found in the initial sample collected 0.25 hr post-dosing. The concentration of 14C in the plasma then decreased in what appeared to be a triexponential manner. A 5-fold decrease in plasma radioactivity occurred during the first 4 hr post-dosing. After 24 hr, the plasma 14C concentration was only 3% of peak level, and by 72 hr the plasma contained only 1.3% of the peak concentration.

Half-lives estimated for the rapid initial, middle and terminal phases were 0.76, 3.6 and 38.5 hr. The area under the plasma 14C-concentration time curve (AUC), volume of distribution (Vd) and whole body (CLT) and renal clearance (CLr) were calculated as 24.03 hr ug equiv 14C-TIPA/g plasma, 1.8 l/kg body weight and 7.77 and 6.51 ml/min/kg body weight, respectively.
Details on distribution in tissues:
Less than 1% of the administered dose remained in the carcass and tissues 72 hr post-dosing. Concentrations of radioactivity were low in all tissues with the liver containing the greatest concentration, approximately 0.02% of the dose/g wet weight. The remaining tissues and carcass contained less than 0.01% of the dose/g wet weight. However, due to its greater mass, the majority of the radioactivity was in the carcass.
Details on excretion:
Most of the dose (64-70%) was excreted in the urine during the 0-6 hr collection interval. An additional 9-14% of the radioactivity was excreted in the 6-12 hr interval. By 24 hr post-dosing, 82.8% of the dose was recovered in the urine and only 1% of the dose was excreted in the urine between 24 and 72 hr post-dosing. The excretion of radioactivity in the urine was even faster than predicted by the plasma 14C-data. Normally the fraction of the dose excreted during an interval will be proportional to the AUC for the material in the plasma for that interval versus the AUC for the material in the plasma from time 0 to infinity. In this case, 98.8% of the radioactivity in the urine was excreted within 24 hr. However, the AUC for radioactivity in the plasma for the 0 to 24 hr interval represented only 68.9% of AUC for the 0 to infinity interval (i.e., 16.56 versus 24.03 hr µg equiv 14C-TIPA/g plasma). Thus, the potential for TIPA to accumulate is even less than predicted by the plasma 14C-data.

As with the urine, most (84.6%) of the fecal radioactivity was eliminated during the first 24-hr post-dosing.

The amount of radioactivity in the 0-12 hr collection interval (3-4% of the dose) represented over 86% of the 14CO2 excreted during the entire 72 hr post-dosing interval.

GC/MS analysis of the pooled urine specimens indicated that virtually all radioactivity in the urine represented unchanged 14C-TIPA. Mass spectra and extracted ion chromatograms for the derivatized urine extract and TIPA standard were identical. Only a single peak, with the same retention time as derivatized TIPA standard, was observed in the GC/MS/Radiogas chromatogram of the derivatized urine extract. Additionally, 95 and 80% of the radioactivity in the 0-6 and 6-12 hr pooled urine sample, respectively, could be accounted for by the amount of TIPA found in these samples by GC/MS.
Toxicokinetic parametersopen allclose all
Toxicokinetic parameters:
half-life 1st: 0.76 hr in plasma
Toxicokinetic parameters:
half-life 2nd: 3.6 hr in plasma
Toxicokinetic parameters:
half-life 3rd: 38.5 hr in plasma
Toxicokinetic parameters:
AUC: 24.03 hr ug equiv 14C-TIPA/g plasma

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
GC/MS analysis of the pooled urine specimens indicated that virtually all radioactivity in the urine represented unchanged 14C-TIPA. Mass spectra and extracted ion chromatograms for the derivatised urine extract and TIPA standard were identical. Only a single peak, with the same retention time as derivatised TIPA standard, was observed in the GC/MS/Radiogas chromatogram of the derivatised urine extract. Additionally, 95 and 80% of the radioactivity in the 0-6 and 6-12 hr pooled urine sample, respectively, could be accounted for by the amount of TIPA found in these samples by GC/MS.

Any other information on results incl. tables

- No signs of toxicity were noted during the in-life phase of the study. The doses delivered were within 7-12% of the targeted dose levels. Between 94% and 96% of the administered radioactivity was recovered in the urine, feces, 14CO2, tissues / carcass, and final cage wash.

- The principle route of excretion was urine, which contained 81-85% of the total dose. Feces contained 4-7%, 3-5% was eliminated as 14CO2, <2% was recovered in the tissues/carcass and final cage wash. The amount of 14C in the traps for volatile organics was negligible.

- Highest concentration of radioactivity was found in the initial blood sample 0.25 hours post-dose. The concentration decreased in a triexponential manner. A 5-fold decrease in radioactivity was accomplished in the first 4-hour post-dosing.

- Half-lives for the rapid initial, middle, and terminal phases were 0.76, 3.6, and 38.5 hours, respectively. Most of the dose of radioactivity (64-70%) was excreted in the urine 0-6 hours post-dosing. An additional 9-14% was excreted in the 6-12 hour interval. By 24 hours post-dosing, 82.8% of the dose was excreted. As with urine, 84.6% of the fecal radioactivity was eliminated during the first 24 hours.

Applicant's summary and conclusion

Conclusions:
- Orally-administered TIPA was rapidly and extensively absorbed by the rat. Based on the amount of radioactivity in urine and cage wash, a minimum of 83.8% of the orally-administered TIPA was absorbed.
- GC/MS analysis of pooled urine indicated that virtually all radioactivity in the urine represented unchanged 14C-TIPA, and was rapidly excreted.
- Based on its rapid elimination, TIPA is not assumed to accumulate in the rat upon daily administration. The data suggest that the excretion of accompanying 2,4-D was not affected by co-administration with TIPA.
Executive summary:

This study examined the metabolism and excretion of triisopropanolamine-1-14C (14C-TIPA) in male rats when administered concomitantly with 2,4 -dichlorophenoxyacetic acid (2,4 -D), and was conducted to support re-registration of products containing the TIPA salt of 2,4 -D (2,4 -D TIPA).

Four male Fischer 344 rats were given a single oral dose of a solution providing targeted doses of 10 mg 2,4 -D/kg and 10.7 mg 14C-TIPA/kg of body weight.

The concentration of radioactivity in the plasma peaked 0.25 hr post-dosing at 4.48 +/- 1.19 ug eq/g plasma and then decreased in a tri-exponential manner. Between 94 and 96% of the administered radioactivity was recovered in the urine, feces, expired 14CO2, tissues/carcass and final cage wash. The major route of excretion was the urine with approximately 80% of the dose excreted by this route in the first 24 hr post-dosing and 81 to 85% excreted by 72 hr post-dosing. The feces accounted for only 4 to 7 % of the dose. Expired 14CO2 accounted for 3 to 5% and the final cage wash ~1% of the dose. Less than 1% of the administered radioactivity remained in the tissues and carcass when these rats were sacrificed 72 hr post-dosing. Essentially all radioactivity excreted in the urine represented unchanged 14C-TIPA based on GC/MS and GC/MS/radiogas analysis of the urine excreted 0 -12 hr post-dosing. Additionally, the urinary excretion of 2,4 -D during the 0 -12 hr post-dosing interval (70.5% of the dose) was nearly identical to that excreted during this interval following oral administration of a 1 mg 2,4 -D/kg dose (69.3 +/- 13.1%).

These data demonstrate that orally administered 14C-TIPA was rapidly absorbed and rapidly excreted primarily in the urine as unchanged TIPA. Due to its rapid elimination, 14C-TIPA should not accumulate in the rat upon daily administration.