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Basic toxicokinetics

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Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard protocol toxicokinetic study conducted according to GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): AMP

- Substance type: Organic (amino alcohol)
- Physical state: Liquid
- Analytical purity: Radiolabelled = 99.0% pure
ANGUS Chemie, GmbH, Ibbenburen, Germany (Batch # TA074801Z1; Lot # 66545-00264239)
(Order #IB-89-05; Certificate of Analysis, ANGUS Chemical Company, Ibbenburen, Germany, 2005)
- Radiochemical purity (if radiolabelling): 99.0%
- Specific activity (if radiolabelling): 22.98 mCi/mmol
- Locations of the label (if radiolabelling): 1-14C

- Stability under test conditions: Stable
Radiolabelling:
yes
Remarks:
C14 AMP

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
- Species and Sex: Rats (male and female)
- Age at Study Start: ~ 8-10 weeks (~200 g body weight)
- Strain and Justification: Fischer 344 rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, the reliability of the commercial supplier, and because the rat is the preferred species for pharmacokinetic/metabolism studies.
- Supplier and Location: Jugular vein cannulated rats were obtained from Taconic Inc. (Germantown, New York). Non-cannulated animals were obtained from Charles River Laboratories Inc. (Raleigh, North Carolina).
- Physical and Acclimation: Upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International), each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes. Non-cannulated animals were housed two per cage in stainless steel cages and acclimated to the laboratoryenvironment for at least one week prior to use, including at least two days in metabolism cages. The stainless steel cages had wire-mesh floors and were suspended above catch pans. Cages contained a hanging feeder and a pressure activated lixit valve type watering system. Jugular vein cannulated rats (surgery performed by the supplier) were acclimated in metabolism cages for approximately four days prior to use. Rodent jackets (Alice King Chatham, Los Angeles, California) were placed on the animals dosed dermally to protect the application site from grooming. The dermally dosed animals were acclimated with rodent jackets following a scheduled regimen (2 hours on day-1 and 4 hours on day-2 followed by 24 hours/day from day-3 until sacrifice).

- Housing: Following administration of the test material, the animals were housed one per cage in glass Roth-type metabolism cages, which are designed for the separation and collection of urine, feces, CO2, and organic volatiles. Air was drawn through the metabolism cages at ~ 500 ml/minute.
- Randomization and Identification: Animals were selected from those available having acceptable patent jugular veincannulae and randomly assigned to treatment groups using a computer-drivenrandomization procedure and identified by a uniquely assigned numbered metal eartag.
- Feed and Water: Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water were provided ad libitum during the pre-exposure and study periods, except that feed was withdrawn from all orally dosed animals approximately 16 hours prior to the administration of 14C-CS-1135 and was returned about four hours post-dosing. Analysis of the feed was performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source is periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants are conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting.

- Animal Welfare: In response to the Final Rules amending the U.S. Animal Welfare Act promulgated by the U.S. Department of Agriculture effective October 30, 1989, the Animal Care and Use Activities (ACUA) required for the conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC has assigned File Nos. Metabolism 01 and Animal ID 01 to these Animal Care and Use Activities.

Administration / exposure

Route of administration:
other: Oral and Dermal
Vehicle:
water
Details on exposure:
Oral route:
Rats recieved a single oral gavage dose of 17.7 mg/kg bw AMP in water.
Dose was prepared immediately prior to dosing and stability in vehicle was confirmed to be >2 weeks
The target dose volume was 5 g/kg and a target radioactivity level of 125 μCi/kg BW.

All orally dosed animals were fasted overnight through approximately four hours post-dosing. Animals receiving dermal application of the test materials were not fasted prior to dosing.

Dermal Route:
Dose solution was prepared in water at the target concentration of ~29 mg/ml and applied at a dose volume of 10 μl/cm2 to 12 cm2 rat skin, which
resulted in a dose of ~3.5 mg AMP (120 μl of dose solution) to ~200 g rat resulting in the dose of ~17.5 mg/kg. Dermal dose solution contained ~2.6 mg/ml (~833 μCi/ml) radiolabeled AMP and ~26.5 mg/ml non-radiolabeled AMP (~100 μCi per rat or ~500 μCi/kg).
Dose Site Preparation
Animals were anesthetized with isoflurane and the hair on the back of each rat was clipped approximately 18 hours prior to dosing. Access to the dose site was restricted by the use of a rodent jacket. The frames were attached 18 hours prior to dosing.
Protective Appliance
The ~1.5 mm thick Teflon frames (4 cm × 5 cm with a 3 cm × 4 cm cut-out opening), shaped into a saddle, were positioned intrascapularly as far anteriorly as possible and attached to the animal using Permabond Industrial Grade 910 adhesive (National Starch and Chemical Co., Englewood, New Jersey). In addition, each rat was fitted with a rodent jacket (Lomir Biomedical, Inc., Malone, New York) to prevent the animals from grooming the dose site. Dose Application
Animals were anesthetized again with isoflurane for dosing. A measured dose of 14C- AMP was applied topically using a round tipped feeding needle attached to an all glass syringe to an approximately 12 cm2 area. The dose solution was applied evenly to the skin in a volume of 10 μl/cm2. The syringe and feeding needle used for application of the test materials was weighed to determine the actual dose applied.
Site Protection
The dose site was semi-occluded by covering with Teflon Spectra/Mesh macroporous filter material (Spectrum Laboratories, Inc., Rancho Dominguez, California), which was attached to the Teflon frame using a Velcro fastner to prevent the animal from grooming the dose site. Test animal access to the dose site was restricted by use of the rodent jacket.

Due to some degree of volatility when applying the test material dermally, the air from these cages was trapped and assessed for radioactivity in order to get a more complete mass balance
Duration and frequency of treatment / exposure:
Single oral or dermal exposure
Doses / concentrations
Remarks:
Doses / Concentrations:
Oral - 17.7 mg/kg bw
Dermal - 17.5 mg/kg bw
No. of animals per sex per dose:
4 males per dose route.
Control animals:
no
Positive control:
No
Details on study design:
This study consisted of a blood/plasma 14C concentration-time course (after oral and dermal application) to determine peak (Cmax) blood/plasma 14C concentrations and ADME experiments to determine absorption (including penetration of AMP from skin), distribution, metabolism, and elimination of AMP following either oral or dermal application. The study continued until ≥ 95% of the administered dose was recovered in the excreta and/or up to seven days post-dosing. Blood sampling from the dermally dosed animals was continued for 7 days regardless of the recovery of the administered dose in order to determine any post-application penetration of radioactivity through the skin at the application site.

The rats dosed orally were fitted with indwelling jugular vein cannulae which were used to collect blood for the determination of time course 14C concentration of the test materials in plasma and red blood cell (RBC). The time course concentration data was used to determine peak (Cmax) and halfpeak (½ Cmax) plasma 14CAMP concentrations. Approximately 0.2-ml blood was collected at chosen times (0.25, 0.5, 1, 2, 4, 8, 12, 24 hours, and every 24 hours thereafter or until the jugular vein cannulae remained patent) and plasma prepared by centrifugation. Weighted aliquots (~25μl) of whole blood were placed in pre-weighed vials containing extraction and stabilizing solvents and stored at –80oC for the analysis of AMP.

Aliquots of RBC were oxidized as described below and the RBC and aliquots of plasma analyzed for radioactivity by liquid scintillation spectrometry (LSS). Sampling of blood from the cannulae was stopped when radioactivity decreased to below the limit of detection, as described below, from all groups with the exception of dermally dosed animals. Samples collected from dermally dosed animal were counted for a longer period of time (i.e., 2 hours/sample) once the radioactivity became below twice the background levels in order to confirm that extremely low levels of the dermally applied radioactivity was not moving into the blood stream from the deeper layers of skin. This, along with collection of samples for 7 days instead of terminating the study upon recovery of ≥95% of the dermally applied dose, was done to confirm that any radioactivity associated with the skin was not becoming systemically bioavailable.
Details on dosing and sampling:
Oral and Dermal dosed rats:
Time course concentration of AMP in whole blood (Plasma and RBC's) was assessed - blood taken via the cannula at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours, and every 24 hours thereafter or until the jugular vein cannulae remained patent.

Dermal:
Animals dosed dermally were anesthetized with isoflurane six hours post-dosing. The macroporous filter material covering was removed and the skin at the dose site was washed five times with cotton tip applicators dipped in an aqueous solution of detergent (i.e., ~2-4% Ivory dish washing liquid, Proctor and Gamble Co., Cincinnati, Ohio). The dose site was also rinsed several times with a gauze soaked with water and the area blotted dry with gauze squares. Following washing, the Teflon Spectra/Mesh covering was replaced and returned to their cages for the continuous collection of time-course blood, urine, feces, and terminal collection of tissues. Radioactivity was determined in the Teflon Spectra/Mesh coverings, the cotton tips, and gauze to determine the dose remained at the site six hours after the application.

During the first 24 hours after the application of 14C-AMP to the skin, the air was drawn through a charcoal trap to trap the test material(s) volatilizing from the application site. This was conducted since the test material was found to be evaporating from the vehicle. Charcoal traps were used to trap the volatile test materials from the site for the first 24 hours as described above.

Urine
All urine voided during the study was collected in dry-ice cooled traps. The urine traps were changed at 12 and 24 hours post-dosing, followed by 24-hour intervals for the remainder of the study. 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 analyzed for radioactivity by LSS as described below. Equal volume aliquots of urine samples from the 0–12 hour and 12–24 hour collection intervals was pooled and stored at -80ºC for chemical analysis.

Feces
Feces were collected in dry-ice chilled containers at 24-hour intervals. An aqueous homogenate (~ 25% w/w) was prepared and weighed aliquots of these homogenates oxidized (OxiMate 80 Sample Oxidizer, PerkinElmer Life Sciences, Inc., Boston, Massachusetts) and quantitated for radioactivity by LSS. In addition, equal volume aliquots of fecal homogenates from each animal was taken from the 0–24 hour collection interval and pooled. These pooled samples were stored at –80ºC for possible chemical analysis.

Expired Volatiles
Air was drawn through the cages at approximately 500 ml/minute. Upon exiting each cage, the air was passed through charcoal to trap organic volatiles. The charcoal traps were changed at 24 hours. Radioactivity trapped on the charcoal was desorbed with weighed amounts of toluene. Weighed aliquots of the toluene were analyzed for radioactivity. Less than 1% of the administered dose was detected in the charcoal trap of the first orally dosed group during the first 24 hours of trapping. Therefore, the replacement traps were not analyzed for radioactivity and air was not trapped for any other groups. For animals that were exposed dermally to the test materials, a probe study was conducted to determine the volatility of the dermally applied 14C-AMP during the exposure. Radioactivity in the charcoal extracts was 3 to >70 fold higher than background; therefore, expired air was trapped for the first 24 hours post-dermal application animals.

Expired CO2
Following the charcoal trap (described above) the expired air was passed through a solution of monoethanolamine:1-methoxy-2-propanol (3:7 v/v) to trap expired CO2 and analyzed for radioactivity. The CO2 trap was planned to change at 12- hour intervals through 48 hours followed by 24-hour intervals for the remainder of the study. However, < 1% of the administered dose was detected in the CO2 trap in the first orally dosed group during the first 12 hour interval therefore, CO2 traps were not used in the remainder of the study.
Statistics:
Descriptive statistics used (e.g. mean +/- SD)

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
AMP was rapidly absorbed from the Gastrointestinal tract following oral dosing, Cmax reached in less than 0.3 h. Almost the entire orally administered dose was absorbed

Dermal Dosing:
Total recovery of applied radioactivity was approximately 82%.
A total of approximately 40% of the administered dose was considered unabsorbed
- skin wash 28%, application site covering 9% and charcoal trap 2%
The total dermal absorption of AMP was approximately 43% of the administered dose.
Details on distribution in tissues:
Oral dose:
at study termination, 2.2% of the dose was found inthe carcas, 0.8% in the liver and 0.3 in the skin.
AMP partitioned equally between Red Blood Cells and Plasma (4.45 microg/g in each at Cmax)


Dermal Dose:
AMP in Red Blood Cells was 40% higher than inplasma (0.15 microg/g vs 0.09 microg/g)
8% of the dose was found in the dose site skin at the end of the study (168 hours), of this, 0.4% of the radioactivity was found in the stratum corneum
6% of the dose was found in tissues, of this about 5% was found in skin (not dose site) and 0.3% was found in the liver.
Details on excretion:
Oral Dose:
Urinary elimintation of AMP accounted for approximately 91% of the adminsitered dose. Most of this dose (74%) was eliminated in the first 48 hours. Fecal elimination accounted for approximately 6% of the administered dose. At the end of the study (168 hours post dosing) only approximately 4% of the dose remained in tissues.
There was a biphasic elimination of AMP. The first phase was rapid and lasted for about 6 hours post dosing, followed by the second phase which was slower with a half life of approximately 41 hours (plasma) and 69 hours (RBC) indicating that AMP appears to sequester into the RBC's.

Dermal Dosing:.
Urinary excretion appears to have accounted for approximately 56% of the absorbed dose, and fecal elimination accounting for approximately 2%. Of the dose excreted in the urine, the majority (approx 5%) was recovered within 48 hours of dosing.
2% of the dose was found in the charcoal filter indicating some excretion via exhalation.
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
Tmax: 0.3 hours (RBC and Plasma, oral dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 4.45 microgram/g in plasma (oral dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 4.45 microgram/g in RBC (oral dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 41 hours (plasma, oral dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 69 hours (RBC, oral dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 29.7 microgram hours/g (plasma, oral dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 34.2 microgram hours/g (RBC, oral dose)
Test no.:
#1
Toxicokinetic parameters:
Tmax: 4 hours (RBC, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
Tmax: 8 hours (Plasma, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.09 microgram/g in plasma (dermal dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.15 microgram/g in RBC (dermal dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 38 h (plasma, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 57 hours (RBC, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 3.7 microgram hours/g (plasma, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 5.4 microgram hours/g (RBC, dermal dose)

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
AMP does not appear to be metabolised.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
AMP is almost completely absorbed following adminstration of an oral bolus dose. Almost 100% of the dose had been excreted via the urine and feces within the course of the study (168 hours), the vast majority of the dose excreted within the first 24 hours. It is not metabolised prior to excretion and does not appear to accumulate in any tissues.

Dermal dosing led to an absorption of approximately 40% of the dose, the majority of which was excreted in the urine. Of the absorbed dose , approximately a quarter remained in the skin at the dose site and it is not clear if this would subsequently have become systemically available.

Based on this data there is no concern that AMP would accumulate in the body.
Executive summary:

Groups of rats were orally or dermally administered 14C-CS-1135 or 14C-AMP and absorption, distribution, metabolism and elimination determined. The orally administered CS-1135 was rapidly absorbed reaching Cmax within one hour and eliminated rapidly in urine. The rapid absorption was apparent from the drop in the dose from the GI tract within three hours between Cmax (56±10 to 68±9%) and ½Cmax (13±3 to 16±6%). Total urinary elimination accounted for 91-99% of the dose, most (77-92%) occurring within the first 48 hours. Fecal elimination accounted for only 4-8% of the dose. Absorption, distribution, and elimination of AMP was almost identical to CS-1135 with relatively faster absorption, reaching Cmax in <0.5 hour. Only 3-4% of the dose was found in tissues at the end of the study. Elimination of radioactivity from blood was biphasic with a rapid distributional (α) phase lasting for 24 hours for CS-1135 and 6 hours for AMP, followed by a slower elimination phase (β). The plasma t½ of the β phase of CS- 1135 was between 20 and 39 hr; females were slightly more efficient in elimination. The RBC t½ of the β phase of CS-1135 was slower (42-55 hours) indicating possible sequestration of radioactivity in RBCs. The administered CS-1135 was rapidly metabolized, apparently prior to reaching the systemic blood circulation. Out of 176 samples, parent CS-1135 was detected in only 10 samples without any specific pattern. AMP, one of the major metabolites of CS-1135, was detected in most of the blood samples. The AUC0→∝ of radioactivity in plasma and RBC was within dose proportionality between the low and high dose. The time-course decline of AMP derived radioactivity from plasma and RBC was almost identical to CS-1135 with slightly longer β elimination half-life. AMP was detected in the blood collected from the rats dosed with AMP at quite consistent concentrations found in rats dosed at an equimolar concentration of CS- 1135. Only the first three samples had ~2-fold higher AMP than was observed in CS-1135 dosed rats. There was an indication of sequestration of AMP to RBC. The majority (>80% in urine and ~8% in feces) of the orally administered radioactivity recovered in excreta from 14CCS- 1135 dosed rats was AMP and only one metabolite above 5% (6-7% in urine) of the administered dose. Urine of the 14C-AMP dosed rats contained only the parent compound.

Most of the dermally applied 14C-CS-1135 evaporated rapidly from the application site and recovered in the charcoal trap. Very little radioactivity was found in the dose site skin, averaging ≤1% of the dose with only ≤0.05% of the dose found in the stratum corneum. The total dermal absorption of 14C-CS-1135 was 13-15% of the dose. Out of which, ~11% was eliminated in urine, most (~8%) within 48 hours. Fecal elimination comprised <0.5% of the dose. The Cmax was reached within one hour post-dosing indicating rapid penetration. The difference in the AUC0→∝ between the two routes was 11 to 14-fold, consistent with the 13-15% dermal absorption compared to ~95% oral absorption. The AUC0→∝ of the dermally administered dose in RBC was 56-90% higher than plasma suggesting sequestration in RBCs. The elimination of radioactivity from blood was biphasic with a relatively slower α phase due to the presence of dose at the application site for six hours and its continuous penetration for some time after the removal/washing. The slope of the β phase was almost parallel to the oral group resulting in almost the same t½β for most of the rats. The total dermal absorption of 14C-AMP was ~43% of the dose. Twenty four percent of the total dose was eliminated in urine, most (~18%) within 48 hours. Fecal elimination was ~2%. Around 6% of the dose was found in tissues. It took much longer to reach Cmax after dermal application (~4 hours) than the oral dose (0.3 hour), indicating slower dermal penetration of AMP. The elimination of the radioactivity from blood was biphasic with relatively slower α phase. The t½β for most of the rats was similar between the two routes. The dermally applied radioactivity was eliminated slower (49% longer t½) from the RBCs than plasma, resulting in 44% higher AUC0→∝, an indication of sequestration of 14C-AMP derived radioactivity in RBCs, similar to that observed in the orally dosed group. Only AMP was detected in the urine of animals dosed dermally with 14C-CS-1135, accounted for 7-9% of the administered dose. Only AMP was detected in the urine of the rats dosed dermally with 14CAMP.

CS-1135 was rapidly hydrolyzed in whole blood, plasma and urine. The level of CS-1135 became undetectable in blood and plasma within 15 minutes after fortification to control samples. Similarly, CS-1135 was rapidly hydrolyzed in urine at room temperature.