2,6-dibromo-4-cyanophenyl heptanoate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 May - 20 Dec 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dibromo-4-cyanophenyl heptanoate

EC Number:

260-300-4

EC Name:

2,6-dibromo-4-cyanophenyl heptanoate

Cas Number:

56634-95-8

Molecular formula:

C14H15Br2NO2

IUPAC Name:

2,6-dibromo-4-cyanophenyl heptanoate

Radiolabelling:

yes

Remarks:

The test substance was labelled with 14C in the aromatic ring (u-14C phenyl)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa-Credo, France
- Age at study initiation: young adults
- Weight at study initiation: 155 - 264 g (males), 165 - 221 (females)
- Housing: metal, wire-mesh bottomed cages before study, then individual metabolism units for single high and low dose exposure. Animals used for the repeated low dose exposure were housed in groups of up to 5 in wire mesh bottomed cages. After the 14th dose, five animals were transferred to individual metabolism units.
- Diet: conventional lab animal diet, ad libitum
- Water: tap water, ad libitum
- Fasting period: 16 h before (diet only)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 ± 1.5
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: polyethylene 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each dose solution was prepared on the day of dosing and samples taken before, during and after dose administration were analytically assayed for correct concentration of active ingredient.

Duration and frequency of treatment / exposure:

single treatment or repeated dose for 15 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

For details, please refer to "Details on study design".

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

For details, please refer to "Details on study design".

No. of animals per sex per dose / concentration:

basically 5 (for details, please refer to "details on study design")

Control animals:

no

Positive control reference chemical:

None

Details on study design:

The study consisted of a series of 6 experiments, including 5 based on single oral administration and one repeated dose experiment as follows:
Experiment/group 1: orally administered single dose of 20 mg/kg bw to 5 males and 15 females for analysis of biokinetics and adsorption, distribution, metabolism and excretion (ADME)
Experiment/group 2: orally administered single dose of 2 mg/kg bw to 5 males and 5 females for ADME analysis
Experiment/group 3: orally administered repeated doses of 2 mg/kg bw to 8 males and 8 females for 15 days for ADME analysis (only 5 animals per sex were used for excretion and metabolism)
Experiment/group 4: orally administered single dose of 20 mg/kg bw to 5 males and 5 females for blood pharmacokinetic data
Experiment/group 5: orally administered single dose of 2 mg/kg bw to 5 males and 5 females for blood pharmacokinetic data
Experiment/group 6: orally administered single dose of 20 mg/kg bw to 6 females for analyzing skin and fur

The repeated dose group was administered non-radiolabelled test substance for 14 days followed by a final administration of a single radiolabelled dose as described for the other study groups.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
In general, the following tissues and body fluids were sampled:
urine, feces, blood (separated into plasma and erythrocytes), organs and tissues (liver, kidney, heart, lungs, brain, fat, skeletal muscle, adrenals, spleen, bone and marrow, gastro-intestinal tract with contents, ovaries, testes, uterus and thyroid) residual carcass, skin and fur

Samples taken from the specific experiments were as follows:
Experiment/group 1: urine, feces, blood, organs, tissues and residual carcass. The tissues taken from the additional female rats for the biokinetic study at 24 and 72 h following administration were liver, kidney, skeletal muscle, thyroid, cardiac blood and the skin and fur.
Experiment/group 2: urine, feces, blood, organs, tissues, residual carcass, skin and fur
Experiment/group 3: urine, feces, blood, organs, tissues, residual carcass, skin and fur
Experiment/group 4: blood for analyzing pharmacokinetic data
Experiment/group 5: blood for analyzing pharmacokinetic data
Experiment/group 6: skin and fur

- Time and frequency of sampling:
Urine was sampled in intervals of 24 h
Feces was sampled in periods of 24 h
Carcass, organs and tissues were sampled at necropsy.
Blood for pharmacokinetics (Group 4 and 5): 0.25, 0.5,1, 2, 3, 5, 7, 8 and 24 h after dosing and at twenty-four hour intervals thereafter until approximately 336 h post-dosing
Skin and fur: at necropsy (only Group 1)

MEASUREMENT OF RADIOACTIVITY AND USED ANALYTICAL METHODS
The following techniques for measuring radioactivity and for identification of substances were used in the present study:

1) For the measurement of radioactivity liquid scintillation counting (LSC) was used.
LSC
Measurement of liquid samples:
The following liquid scintillation counters used:
Packard 1900 TR liquid scintillation counter
Cocktail: Ultima Gold liquid scintillation cocktail

Measurement of solid samples:
Feces were homogenized with methanol, centrifuged and the supernatant collected. After resuspension of the residue, this step was repeated twice. All supernatants were pooled and assayed as described for liquid samples. The solid material was air-dried (30 °C) and grounded to a uniform powder. Samples were weighed and combusted in an oxygen atmosphere (Packard Model 306 or 387 Tri-Carb). Scissor-minced portions of fat, testes, bone and marrow, uterus and ovaries, and of the adrenals were combusted directly (0.01 g to 0.3 g). Cardiac blood samples from Group 2 and 3 were also combusted directly after drying of weighed aliquots (0.2 g) on Combusto-pads contained in Combusto-cones (supplied by Packard Instruments SA, Rungis, France). Other tissues were homogenized, if necessary, HPLC grade water was added. The carcasses were also homogenized with the addition of water. Portions (0.15 g to 0.30 g) of the homogenates were weighed into combusto-cones and then combusted using the sample oxidizer (Packard Model 306 or 387) following the addition of a small quantity of cellulose powder (0.05 g to 0.1 g). For all samples, Carbosorb was used to trap the combustion product carbon dioxide. which was then mixed with an appropriate scintillation cocktail (Permafluor, 12 mL) prior to radioassay.

Blood samples from Group 1, 4 and 5 (up to 0.17 g) were added to a mixture of solubilizer (Soluene) and isopropanol (1:1 v/v; 1 mL), when necessary the mixture was warmed to aid homogenization. The Hionic Fluor liquid scintillation cocktail (10 mL) was used.

The following liquid scintillation counters used:
Packard 1900 TR liquid scintillation counter
Cocktail: Ultima Gold liquid scintillation cocktail
Limit of detection/quantification (LSC):
Not reported

2) Metabolism investigations
High Performance Lqiuid Chromatography (HPLC) and Thin Layer Chromatography (TLC)
Individual and pooled urine and individual and pooled fecal extract samples were analyzed by HPLC and TLC. The mobile phases was composed of A = Methanol/glacial acetic acid (0.1% v/v) and B = Water/glacial acetic acid (0.1% v/v). Injection volumes were up to 150 µL and Kratos Spectroflow 783 Spectrophotometer and IN/US fi-RAM Radioactivity Monitor with 400 µL calcium fluoride flow cell were used as detectors. Data was processed IBM-compatible personal computer equipped with the LabChrom Measuring/Evaluation software. For TLC glass-backed precoated silica gel plates containing a fluorescent indicator (254 nm) were used. The solvent system (A) used was: Toluene/Acetone/Dichloromethane/glacial acetic acid, (50:25:25:1 by volume). A further solvent system (B) was used to obtain an improved separation (Hexane/Glacial acetic acid/Diethyl ether, (75:15:10 by volume)). Radioassay was performed using the AMBIS detector.

Urine samples:
Samples were taken from each individual animal at each time point up to 96 h post-dosing for the males and 120 h post-dosing for the females for all three ADME experiments. Further samples were pooled according to sex and time period for metabolite quantification and identification.
Time intervals: 0-24 h, 24-48 h, 48-72 h and 72-96 h (both males and females) and 96 - 120 h (females)
Moreover, aliquots of pooled urine (0.5 mL) from males and females from the high dose group (group 1) were used for analysis of enzymatic deconjugation.
Feces samples: pooled according to sex for 0 – 24 h and 48 – 168 h for both males and females of the high dose group (1), 0-168 hour pooled samples for the two low dose groups (group 2 and 5), all samples were concentrated.

Methanolic extracts from selective tissues:
Methanolic extracts were prepared from the thyroids, liver, kidney, muscle, plasma samples, skin and fur. Skin and fur were frozen in liquid nitrogen, milled for homogenization, and then extracted with methanol.

Identification of metabolites:
Metabolites were identified using authentic standards.

Limit of detection/quantification (HPLC):
Not reported

Statistics:

The calculation of % dose recovery and compound concentrations, including means and standard deviations, were carried out by use of the Microsoft Excel (version 4.0) spreadsheet program on an Apple Macintosh Ilsi computer. It is possible that in certain tables the presented means do not calculate exactly from the presented individual data. This is due to rounding-up differences experienced from the use of the spreadsheet program.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

For both, male and female rats treated by single or repeated dosing, the test material was rapidly and extensively absorbed from the gastrointestinal tract, as evidenced by the high excretion level mainly involving the urine.

Main ADME results 2

Type:

distribution

Results:

Maximum plasma levels were measured after 24 h and declined thereafter. Radioactivity was distributed to the organs/tissues mainly liver, kidney and thyroid.

Main ADME results 3

Type:

excretion

Results:

Excretion mainly involved urine (73 - 85% radioactivity recovery), with the majority of the administered radiolabel being eliminated in the first 72 h following dosing for the males and 96 h following dosing for the females.

Main ADME results 4

Type:

metabolism

Results:

The metabolism of 14C-test material was rapid and similar in both, male and female rats; as main metabolite, Bromoxynil phenol accounted for 100% of the dose recovered in the urine.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Recovery
The recoveries were found to range from 91.33% to 105.87% of the nominal doses (mean 98.43%, SD±2.5%).

Absorption:
For both, male and female rats treated by single or repeated dosing, the test material was rapidly and extensively absorbed from the gastrointestinal tract, as evidenced by the high excretion level mainly involving urine (73 - 85% radioactivity recovery) and, to a lesser extent, the feces (ca. 6 to 10%).

Pharmacokinetics:
Regarding blood pharmacokinetics, the mean (± SD) whole blood Cmax value for the
females of the high dose groups was found to be 92.47 ± 18.85 µ/g whereas for the males 64.94 ± 7.8 µg/g was reported. The corresponding mean (± SD) Tmax values were 7.0 ± 1.2 h for the females and 7.4 ± 1.3 h for the males. Thus there was no apparent difference between the sexes for the Tmax values and an indication of higher Cmax values for the females.
For the low dose group, the mean (± SD) Cmax values were 7.86 ± 1.1 µg/g for the males and 8.73 ± 1.6 µg/g for the females. The mean Tmax values were 6.6 ± 1.5 h for the males and 6.2 ± 1.1 h for the females. There was no apparent difference between the sexes. The elimination half-life results from both dose levels were very similar yielding mean values of 57.68 h (high dose males), 55.13 h (high dose females), 47.99 h (low dose males) and 64.97 h (low dose females).

For further details, please refer to Attachment 1.

Details on distribution in tissues:

168 h post administration, the highest radioactivity in females was found in blood and plasma throughout all dose groups (plasma concentration of 6.732 μg/g, 1.135 μg/g and 0.959 μg/g for group 1, 2 and 3, respectively). In males, plasma concentration was lower (0.851, 0.289 and 0.220 for group 1, 2 and 3, respectively).The highest mean concentrations in males were found in liver (0.901 μg/g, group 1 and 0.226 μg/g, group 3), and thyroid (0.316 μg/g, group 2).

For females receiving a single dose of 20 mg/kg bw/day, 24, 48 and 72 h after administration, the highest tissue concentration was found in plasma, followed by whole blood, liver, kidney skin/fur, thyroid and muscle. The highest levels were observed in the samples taken 24 h after dosing which progressively declined in all the sampled tissues through to 168 h post-dosing. The lowest values were found in the brain. These values were obtained for females, only.

Summarized results can be found in Attachment 2.

Transfer into organs

Key result

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was distributed to the peripheral tissues mainly liver and kidney.

Details on excretion:

In all dose groups the elimination of radiolabel via the urine was greater than that via the feces. Females showed urinary excretion of 73.3 - 79.34% and males 82.77 - 85.18%. Elimination via feces was 6.3 - 9.63% (females) and 5.86 - 7.16% (males). Additional 4 - 5% of radioactivity were found in the cage wash. The observed reduced systemic clearance of radioactivity by the female animals when compared with the male animals was reflected in the level of radioactivity found in the tissues at 168 h post-dosing where the levels were found to be up to approximately four times higher.
The majority of the administered radiolabel was eliminated in the first 72 h following dosing for the males and 96 h following dosing for the females in all three dose groups. The observed routes and rates of excretion for the two low dose groups were similar, indicating that there was no time-dependent change in the pharmacokinetics of the test substance e.g. enzyme induction.

Summarized results can be found in Attachment 3.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

half-life 1st: 57.68 ± 2.3 h (males, high dose), 55.13 ± 5.2 h μg/g (females, high dose), 47.99 ± 0.6 h (males, low dose), 64.97 ± 3.1 h (females, low dose)

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

Tmax: 7.4 ± 1.3 h (males, high dose), 7.0 ± 1.2 h μg/g (females, high dose), 6.6 ± 1.5 h (males, low dose), 6.2 ± 1.1 h (females, low dose)

Toxicokinetic parameters 3

Key result

Toxicokinetic parameters:

Cmax: 64.94 ± 7.8 μg/g (males, high dose), 92.47 ± 18.9 μg/g (females, high dose), 7.86 ± 1.1 μg/g (males, low dose), 8.73 ± 1.6 μg/g (females, low dose)

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Urine
Urine was analyzed for males up to 96 h post-dosing and 120 h for females. 4 metabolites were detected in urine, named UMET/1 - UMET/4 with increasing retention time. UMET/1 was found to be the major metabolite for males of the high dose group, while UMET/4 was found more frequently in all female groups and in the low single and repeated dose group in males. UMET/3 was only detected in some groups at specific time points. The parent compound was not found, indicating complete metabolization. UMET/4 was identified as Bromoxynil phenol based on reverse-phase HPLC and normal-phase TLC and comparison with a certified standard. UMET/1 - UMET/3 were characterized as sulphate and glucuronide conjugates which all yielded the fourth metabolite after enzymatic hydrolysis.

Feces
Two compounds were detected in feces (named FMET/1 and FMET/2). FMET/2 was only found after single application and the amount of this metabolite was lower than FMET/1. FMET/1 was identical to UMET/4 and thus, representing Bromoxynil phenol, while FMET/2 was identified as the parent compound.

Tissue extracts
Liver samples contained up to 4 radiolabelled components. The major one was identified as Bromoxynil phenol; the remaining unidentified metabolites accounted for less than 1% at their maximum levels. The kidney, muscle, plasma and skin & fur samples were found to contain a single radioactive component which was identified as the major metabolite Bromoxynil phenol.

Summarized results can be found in Attachment 4. The proposed metabolic pathway can be found in Attachment 5.

Enzymatic activity

Enzymatic activity measured:

not measured

Applicant's summary and conclusion

Conclusions:

The toxicokinetic behavior and metabolism of the test compound was investigated in a GLP-compliant study according to EPA OPP 85-1 (similar to OECD 417). During the study, rats were orally treated with 2 or 20 mg/kg bw of the test substance (either as a single dose or, for the low dose, as repeated doses over 14 +1 days). The study is considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. With the use of radioactive-labelled test material, the present study demonstrated that the test compound was rapidly absorbed from the gastro-intestinal tract and readily distributed to the peripheral tissues, mainly the liver, kidney and thyroid. Thereby, the residual radioactivity in tissues in females was higher than in males (6.38 – 10.80% in females vs 1.62 – 3.83% in males). The major route of elimination was the urine which accounted for approximately 80% of the administered dose in all three dose groups, fecal excretion being relatively minor.
Elimination was relatively protracted for the females who were found to be slower than the males, in lines with the higher tissue levels of radioactivity at 168 h post-dose. The major metabolite recovered in tissues, urine and feces was identified as being bromoxynil. The parent compound was only found in small amounts in feces.