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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1994-03-24 to 1997-04-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted with a structural analogous read-across substance in compliance to GLP and to current guidelines. Please refer to IUCLID section 13 for read-across justification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
Test guideline
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Colorless, odorless crystals
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Ml; Jugular vein cannulated and bile-duct cannulated rats of the same strain were also purchased from the same supplier.
- Age at study initiation: 7-9 week old
- Weight at study initiation: ca. 200 g
- Fasting period before study: 16 hr prior to oral dosing
- Housing: individually
- Individual metabolism cages: yes
- Diet: Certified Purina rat chow, ad libitum
- Water: ad libitum
- Acclimation period: At least three days. Bile duct cannulated animals were used within 24 - 48 hr, and Jugular vain cannulated animals were used within 24 hr, respectively, after receiving.

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 1994 To: March 1995

Administration / exposure

Route of administration:
other: oral and intravenous
Vehicle:
other: oral: DMSO, intravenous: sodium bicarbonate solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For intravenous dosing, the doses were prepared in sodium bicarbonate solution. For oral dosing, the doses were prepared in dimethyl sulfoxide (DMSO) solvent which is the best solvent for dissolving the test item.

VEHICLE:
0.05 M sodium bicarbonate solution (i.v.)
- Amount of vehicle: 120-175 µL per animal

DMSO (p.o.)
- Amount of vehicle: 125-190 µL per animal (low dose)
- Amount of vehicle: 550-800 µL per animal (high dose)
- Lot/batch no.: E37617
- Purity: 99.9%
Duration and frequency of treatment / exposure:
There were two parts in this study:
In part 1 study, which consisted of Groups A, B, C, and D, Phenyl Label was used.
In part 2 study, which consisted of Groups E, F, G, and H, Pyridinyl Label was used.

Groups A and E were intravenously dosed at 1 mg/kg
Groups B and F were orally dosed at 10 mg/kg
Groups C and G were orally dosed at 1,000 mg/kg
Each dosing group consisted of 3 subgroups:
(1) rats killed 72 hr after 14C dose
(2) rats for periodic blood sampling and killed 24 hr after dosing
(3) bile-duct cannulated rats killed 48 hr after dosing.
Urine and feces samples were collected from each subgroup at 7 hr, 24 hr and then each 24 hr until sacrifice. Bile samples from bile-duct cannulated rats were collected periodically for 48 hr.

Groups D and H were orally dosed with unlabeled test item at 10 mg/kg for 14 days followed by a 14C dose at 10 mg/kg.
For the repeated unlabeled groups, only 2 subgroups were used and rats were killed either at 24 hr or 72 hr after dosing.
Doses / concentrations
Remarks:
Doses / Concentrations:
Oral:
Low dose level: 10 mg/kg bw
High Dose Level: 1000 mg/kg bw

Intravenous:
Low dose level: 1 mg/kg bw (Limited by solubility of the test item in available intravenous injection vehicle.)
No. of animals per sex per dose:
Each subgroup consisted of 5 male and 5 female rats.
Control animals:
no
Positive control:
not applicable
Details on study design:
- Dose selection rationale:
Low dose: The selection of 10 mg/kg bw is based on a 90 day rat feeding study which obtained a no-observed-effect-level (NOEL) of 5000 ppm in the diet. The 5000 ppm in the diet is equivalent to 350 mg/kg bw/day for male and 430 mg/kg bw/day for female rats. The low dose of 10 mg/kg bw was selected as a NOEL from this study by oral intubation. The dose level of 1 mg/kg bw was used for intervenous injection due to limited solubility of the test item in available intervenous injection vehicle.
High dose: The selection of 1000 mg/kg bw was based on a 90-day feeding study which obtained a low-effect level (LEL) of 10000 ppm in the diet. At this feeding level, reduced body weigh gain, impaired food utilization, disturbance in hematology and clinical chemistry values and increased foamy macrophage in the lung was observed. The 10000 ppm test item in the diet is equivalent to 629 mg/kg bw/day in male and 890 mg/kg bw/day in female rats. Thus a dose of 1000 mg/kg bw is considered high enough to cause some toxic effect but a non-lethal dose.
Details on dosing and sampling:
Dosing:
Groups A and E (i.v., low dose level): A single dose of 1 mg/kg bw with radioactivity of ca. 25 µCi/rat was given to all animals in this group through intravenous injection via jugular vein. Two types of animals were used in this approach:
1) jugular vain cannulated rats for 72 hr and 24 hr subgroup
2) jugular vein and bile-duct doubly cannulated rats for 48 hr subgroup

Groups B and F (p.o., low dose level): A single dose of 10 mg/kg bw with radioactivity of ca. 25 µCi/rat was given to all animals in this group through gavage dosing. Both normal and bile duct cannulated animals were used:
1) normal rats for 72 hr and 24 hr subgroup
2) bile duct cannulated animals for 48 hr subgroup

Groups C and G (p.o., high dose level): A single dose of 1000 mg/kg bw with radioactivity of ca. 25 µCi/rat was given to all animals in this group through gavage dosing. Both normal and bile duct cannulated animals were used:
1) normal rats for 72 hr and 24 hr
2) bile-duct cannulated animals for 48 hr.

Groups D and H (p.o., repeated low dose level): Only normal rats were used for this approach. All animals in this group received a daily oral dose of unlabeled test item at a level of 10 mg/kg bw/day for 14 days followed by a single dose of 14C label test item at 10 mg/kg bw in DMSO with radioactivity of ca. 25 µCi/rat.

METABOLITE CHARACTERISATION STUDIES
The procedure for the collection of blood, bile, urine, feces and tissue samples were the same irrespective of the dosing regimens and groups of rats.

URINE
Separate Sample Collection and Treatment: Urine samples were collected at 7 hr, 24 hr and then each 24 hr thereafter. Microbial degradation of metabolites was prevented by collecting samples in the presence of 1.0 ml of saturated mercuric chloride solution which was placed in the container prior to each collection. A graduated cylinder (100 mL) was used to determine the volume of urine collected at each collection time period. The urine was filtered through a small glass wool plug filter funnel placed on the mouth of the cylinder. The cage was rinsed with water so as to recover most of the urine sample. This was filtered over the glass wool plug and collected in the cylinder. The cage was rinsed twice more with water and the cage washings were combined with the original urine sample in the cylinder. The total volume of urine was noted and the cylinder capped and shaken to attain sample homogeneity. The sample was radioassayed in duplicate by liquid scintillation counting. In this manner urine samples were collected for each collection time point using the same cylinder for all the urine samples of a given rat. Samples were labeled and stored in a freezer at -10°C in screw-capped plastic test tubes.
After the last sample was collected, the rat was removed from the cage. The cage was washed first with a spray of methanol and then with water. The washings were allowed to drain into the urine container. This was transferred to the measuring cylinder in the manner described above, and labeled as Cage Wash. Aliquots of the Cage Wash were taken and counted in duplicates the same manner as with the urine collections.
Pooling of Urine Samples: The urine samples in screw capped plastic test tubes were stored in the freezer till further processing. When all the urine samples for a given group have been radioassayed, they were pooled as a single sample prior to processing for metabolite identification and quantification. Samples with radioactivity less than about 1% of the dose were not combined with other samples. The urine samples were thawed and pooled into a suitable capacity graduated measuring cylinder. The total volume of the pooled urine was noted. The sample was carefully mixed to attain radioactivity homogeneity. The pooled sample was distributed into amber screw-capped 100 ml bottles, properly labeled and stored in a freezer till sample extraction step was executed .

FECES
Separate Sample Collection and Treatment: Fecal samples were collected at 7 hr, 24 hr and then each 24 hr thereafter. The fecal samples from the collection containers were transferred tared zip lock bags and stored in a freezer at -10°C. At processing, the samples were thawed at room temperature; a small amount of distilled water was added to each sample. The mouth of the bag was closed and the feces was thoroughly mixed to form a homogeneous slurry and weighed on an analytical balance. Aliquots of each such prepared fecal sample were taken in triplicate, weighed, air-dried, and oxidized in the biological oxidizer for quantitation of radioactivity content. The remainder of the fecal samples was stored in the freezer till further processing.
Pooling of Feces Samples: When all the fecal samples for a given group had been radioassayed, they were pooled into a suitably sized tared wide mouthed screw cap plastic bottle as a single sample prior to processing for metabolite identification and quantification. Samples with radioactivity less than 1 % of the dose were not combined. The zip lock bags were thoroughly rinsed with distilled water and the washings were transferred to the same plastic bottle. The feces were homogenized to a thick slurry using a Polytron Model PT 3000 homogenizer (Brinkmann). The probe was rinsed with distilled water. The bottle was capped and shaken gently to homogenize the slurry. The slurry was frozen in a dry-ice acetone mixture and the frozen sample was lyophilized and then weighed. The solid obtained was broken down to a fine powder using a spatula. A known amount of the lyophilized feces from a given sample was oxidized in triplicate to determine the radioactivity in the total dry mass of the pooled sample. The lyophilized fecal samples were then stored in the freezer.

BILE
Separate Sample Collection and Treatment: Bile duct cannulated rats were used for bile collection at 1, 2, 4, 7, 24 and 48 hr. After attachment of the bile collecting system, the rat was placed in the metabolism cage. Around 0.1 ml of saturated mercuric chloride solution was placed in the container prior to each collection to prevent possible microbial degradation of the metabolites. The total volume of each bile collection in the measuring cylinder was noted and shaken to attain sample homogeneity. The same measuring cylinder was used for all collections of a given rat. Aliquots of each sample was radioassayed by liquid scintillation counting in duplicate. The remainders of all bile collections were stored in the freezer separately in screw-capped test tubes.
Pooling of Bile Samples: When all the bile samples for a group had been quantified for radioactivity, they were pooled as a single sample prior to processing for metabolite characterization and identification. Samples with radioactivity less than about 1 % of the dose were not combined with other samples. The samples were thawed at room temperature and pooled into a suitable capacity graduated measuring cylinder. The total volume of the pooled bile was noted. The sample was carefully mixed to attain radioactivity homogeneity. The pooled sample was distributed into a suitable container, properly labeled and stored in a freezer till sample extraction step was executed.

BLOOD
Consecutive blood sampling was conducted through standard tail snipping procedures after dosing at 1 hr, 2 hr, 4 hr and 7 hr. About 10 drops of blood, equal to about 0.1 g, were collected each time from each rat into a pre-weighed oxidation thimble placed in a vial. The vial was capped tightly and immediately after sample collection, and weighed. The lid was then removed, and the sample was placed in the hood to dry. The blood sample was then oxidized and the level of radioactivity was quantified by scintillation counting. In addition to the above described whole blood sample, about 5-6 mL of blood was collected directly into a Vacutainer (13 x 100mm, Becton Dickinson) from each animal through cardie puncture at the time the animals was killed. The sample was inverted gently five times after collection, allowed to clot for 30 mm and then span at 13,000 rpm for 12-13 min on a bench-top centrifuge (Dynac II Centnfuge, Clay Adams). The serum was then separated from the blood cells. Aliquots of the serum and the blood cells were taken, respectively, weighed, air-dried, oxidized, and counted for radioactivity content.

TISSUES
Ten tissue specimens were taken from each experimental animal at sacrifice after blood was taken through cardiac puncture. After the animal's abdominal cavity was opened, the tissues were taken in the order roughly from the lowest radioactivity-containing organ to the highest radioactivity-containing organ: fat gonad, muscle, bone, lung, spleen, heart, kidney, and lastly the liver. The brain was taken thereafter. The tissue specimens were stored in pre-weighed containers. The heart, kidney, and liver were rinsed in deionized distilled water to get rid of blood, and tapped dry on paper towels before putting into the containers. They were capped and weighed. The residual carcasses were kept separately in labeled zip lock bags and stored in a freezer at -10°C.
At processing, the samples were thawed at room temperature. A small portion of each tissue specimen (ca. 0.3 g) was weighed out, oxidized, and radioassayed in duplicate. Carcasses were not radioassayed. The remainders of all tissue samples were stored in the freezer.
Statistics:
no data

Results and discussion

Preliminary studies:
A pilot metabolism study with the test item in rats was conducted and reported. The study indicated that only 33-38% of the administered dose was absorbed regardless of the level of dosing, and was rapidly eliminated from the animal body in 24 hours. Residue level in blood was about 5 times higher than in other tissues, which had about the same residue level except brain. The residue level in brain is significantly lower. The excreted radiocarbon was primarily unchanged test item. The major metabolites found were hydrolytic degradation products.
Main ADME resultsopen allclose all
Type:
absorption
Results:
30-50 % after oral administration
Type:
distribution
Results:
Tissue distribution is low (<1% of administered dose)
Type:
metabolism
Results:
The majority of the test item was excreted unchanged. Only several hydrolytic and hydroxylated metabolites were identified.
Type:
excretion
Results:
The elimination half-life was estimated to be about 6 hours for all groups. The test item is eliminated by feces, urine and bile.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Urinary excretion data indicated that the test item was only partially absorbed by oral dosing. Based on the amount of radiocarbon excreted in urine after oral administration compared to the amount of radiocarbon in urine after i.v. administration it is concluded that 30 to 50% of the oral dose was absorbed by rats and there was no difference in percent absorption between 10 mg/kg and 1000 mg/kg dosings.
Details on distribution in tissues:
Radiocarbon retained in tissues and organs were low. Only about 0.2% of the dose was retained in 10 tissues and blood 24 hr after dosing; and it decreased to about 0.02% at 72 hr. Generally, kidney, liver, spleen and lung had higher residue level than heart, fat, muscle, gonad and bone. Brain had the lowest residue level in all groups administered either phenyl or pyridinyl label.
Radiocarbon concentration in blood was higher in rats administered phenyl label than that administered pyridinyl label. Radiocarbon concentration in blood of rats administered pyenyl label also decreased more slowly than that dosed with pyridinyl label. These results indicated that the phenyl moiety (possibly M2) was conjugated to the blood components.
Details on excretion:
Radiocarbon was steadily eliminated in urine and feces after 14C-labeled test item administration. The elimination pattern followed a one-compartment model. For the intravenous administration (Groups A and E), excretion decreased steadily followed apparent first-order kinetics. For the oral dosing (Groups B, C, D, F, G and H), an absorption phase was followed by the elimination phase which followed apparent first-order kinetics. The elimination half-lives ranged between 5 and 11 hr with an average of 6.3 hr. Thus the test item has a short half-life in rats.
Toxicokinetic parameters
Toxicokinetic parameters:
half-life 1st: 6 hours

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Several hydrolytic products and hydroxylation products were also isolated and identified (see Table 1 below). The cyclized intermediate M5 was found in phenyl and pyridinyl labels in all excreta samples examined. M5 was further hydrolyzed to M2 and M1. A trace amount of M2 was detected in urinary samples from the phenyl label. M1 was detected in urine, feces and bile samples in the pyridinyl label. M1 was hydroxylated on the pyridinyl ring which was converted to a keto form and isolated as M9. M1 was also hydroxylated on the methyl group to form the M10 metabolite. M9 was further hydroxylated on the methyl group and M10 was further hydroxylated on the pyridinyl ring to form a common metabolite M19 which was isolated in trace amount in urine samples. M8 was detected in trace amount in ruine and feces samples for the phenyl label. M8 could be an artifact since it was known that the test item degraded in methanol solution to produce M8. The test item was also directly hydrolyzed to M6.
M9, M10 amd M19 were also found in goat and hen excreta, as well as the terminal residues in com plant. Thus, parts of the test item's metabolic pathways in rat are common in other animal species and in com plant.

Bioaccessibility

Bioaccessibility testing results:
not applicable

Any other information on results incl. tables

Table 1: Hydrolytic and hydroxylation products of the test item

M1

Phthalazinone

M2

3,5-difluoroaniline

M3

Symmetric urea

M5

Carbamoyl phthalazinone

M6

2-Acetyl nicotinic acid

M7

Semicarbazide

M8

Carbamate

M9

2-keto-M1

M10

8-hydroxymethyl-M1

M19

2-keto-8-hydroxymethyl-M1

 

 

- Differences between Male and Female Rats:

It appeared that there was no significant difference in absorption, distribution, metabolism and elimination in male and female rats. Administered radiocarbon was partially absorbed in both sexes. The distribution patterns in various tissues and organs were similar between male and female. The metabolic profiles in urine, feces and bile were also similar between sexes. The test item also rapidly eliminated both in male and female.

 

- Differences between Dose Rates:

There was no significant difference in the percentage absorption between the dose rate of 10 mg/kg bw and 1 000 mg/kg bw.

 

- Difference between Phenyl and Pyridinyl Labels:

Blood radiocarbon concentrations for the phenyl label were higher than those for the pyridinyl label. For example in Group A (phenyl label) males, blood contained 0.048% of the dose 72 hr after i.v. administration as compared to Group E (pyridinyl label) males with 0.004% of the dose. The association of radiocarbon from the phenyl label may be due to the generation of M2 which has the tendency to conjugate with blood component such as hemoglobin.

 

- Difference between Single and Multiple Doses:

There was only a slight difference in radiocarbon elimination rate between single and multiple doses. For example the elimination half-life was 5.5 hr for Group B (single dose) male vs. 6.2 hr for Group D (multiple doses) male. Residue levels in tissues for the single dose were slightly lower than the multiple doses. These data showed that the test item had no tendency to bioaccumulate in animal systems.

 

Applicant's summary and conclusion