2,6-dibromo-4-cyanophenyl octanoate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Sep - 12 Sep 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 1984

Deviations:

no

Principles of method if other than guideline:

No guideline is mentioned in the study report, however, the test conduct is widely compliant to the OECD test guideline 417 as adopted 1984.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dibromo-4-cyanophenyl octanoate

EC Number:

216-885-3

EC Name:

2,6-dibromo-4-cyanophenyl octanoate

Cas Number:

1689-99-2

Molecular formula:

C15H17Br2NO2

IUPAC Name:

2,6-dibromo-4-cyanophenyl octanoate

Radiolabelling:

yes

Remarks:

14C ring radiolabel

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River UK
- Age at study initiation: young adults were used
- Weight at study initiation: males: 190 - 212 g; females: 191 - 208 g
- Housing: battery cages with suspended wire mesh floors
- Diet: Laboratory Diet No. 1 (Spratt's Ltd., Barking, UK) ad libitum
- Water: Tap water, ad libitum
- Acclimation period: not reported
ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: From: 11 May To: 12 Sep 1983

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the low dose level of 2 mg/kg bw, the radiolabelled test material was dissolved in polyethylene glycol 400 at a concentration of ca. 0.4 mg/mL. Each rat was then administered orally 1.0 mL of this solution. For the high dose level of 20 mg/kg bw, the radiolabelled test material was dissolved in polyethylene glycol 400 at a concentration of ca. 4 mg/mL. Each rat was dosed orally with 1.0 mL of this solution. The dose administered to each rat was determined by dispensing two samples of 1.0 mL of the dosing solution into volumetric flasks. The doses were diluted to the appropriate volume with methanol and triplicate or duplicate aliquots then measured for radioactivity.

Duration and frequency of treatment / exposure:

single treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

5

Control animals:

no

Positive control reference chemical:

No

Details on study design:

Male and female rats were treated by single oral administration of radiolabelled test material, at dose levels of 2 and 20 mg/kg bw. In fact, the study consisted of following experiments:
Preliminary test: Excretion and retention of radioactivity, preliminary group with 1 male and 1 female receiving a single oral dose of 2 mg/kg bw of radiolabelled test material;
Group 1: Excretion and retention of radioactivity, low dose group with 5 males and 5 females receiving a single oral dose of 2 mg/kg bw of radiolabelled test material;
Group 2: Excretion and retention of radioactivity, high dose group with 5 males and 5 females receiving a single oral dose of 20 mg/kg bw of radiolabelled test material;
Group 3: Plasma concentrations of radioactivity, low dose group with 5 males and 5 females receiving a single oral dose of 2 mg/kg bw of radiolabelled test material;
Group 4: Plasma concentrations of radioactivity, high dose group with 5 males and 5 females receiving a single oral dose of 20 mg/kg bw of radiolabelled test material;
Group 5: Tissue distribution of radioactivity (whole-body autoradiography), 5 females receiving a single oral dose of 20 mg/kg bw of radiolabelled test material.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, feces, blood (separated into plasma and erythocytes), organs, tissues and residual carcass (for radioactivity measurements in Group 1 and 2 and for whole body audiography in Group 5)
- Time and frequency of sampling:
Urine: 0-8, 8-24, and in 24 h-intervals thereafter until 168 h after dosing (Group 1 and 2)
Feces: in 24 h intervals until 168 h after dosing (Group 1 and 2)
Blood: 0.25, 0.5, 1, 2, 3, 5, 7, 24, 48, 72, 96, 120, 144, 168, 216, 264 and 336 hours after dosing (Group 3 and 4)
Tissues collected for radioactivity measurements: 168 h after dosing, lungs, thyroid,
brain, gonads, heart, kidneys, liver, spleen, gastro-intestinal tract, skin and fur and samples of blood, bone marrow, muscle and fat were removed from the carcass (Group 1 and 2)
Tissues and carcasses for whole-body audiography: 7 hours, 1, 3, 7 and 14 days after administration of the dose (Group 5)

With respect to Group 5 which was intended for whole-body autoradiography, each of the five animals was sacrificed after 7 hours, 1, 3, 7 and 14 days following treatment by asphyxiation with carbon dioxide. 20 micron thick sagittal sections were cut of from several levels through the carcass between ovaries and spinal cord using a cryostat with an electrolinear drive.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, feces

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.
2) For the measurement of radioactivity in tissue sections of animals, whole-body autoradiography was used.
3) For separation and evaluation of compounds high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) with radiodetection was used
4) For identification of compounds mass spectroscopy was used (HPLC/MS/MS)

1) LSC
System used: Philips Liquid Scintillation Analyzer (Model No. PW 4700 or PW 4510; Philips 19.V., Eindhoven, Holland) with automatic external quench correction.
Sample preparation:
Solid samples (carcass and feces) were extracted with methanol. After centrifugation, radioactivity was measured in both the extracts and residues. Skin and fur were digested in 20% aqueous sodium hydroxide and diluted with methanol afterwards. Kidneys, livers, gastro-intestinal tract and its contents) were finely minced and homogenized. Samples of urine, plasma, solvent extracts, skin and fur digests, contents of expired air traps and cage washings were mixed with MI-31 scintillator, (Packard Instrument Ltd., Caversham, U.K.). Samples of tissues and residues of extracted carcasses and feces were combusted in oxygen (Automatic Sample Oxidiser, Model 306, Mk 2, Tri CarbJL/packard Instrument Ltd.) and absorbed into Carbo-Sorb and mixed with Permafluor.
Limit of quantification: twice the background level was considered the limit of quantification

2) Whole-body Autoradiography
The 20 µm sections were mounted on type 810 tape and freeze-dried at -50 °C before placing them in contact with either Kodak DBF2 film (Kodak Ltd., Hemel Hempstead, Hertfordshire) or Singul X/RP medical X-ray film (John Blishen Co. Ltd., London) in light-tight cassettes. The audiographs on Kadak DEF2 films were exposed for 21 days at -20 °C and then developed in DX-80 developer and fixed in FX-40 X-ray liquid fixer (Kodak Ltd.). Contact prints were prepared on Ilfospeed grade 2 semi-matt paper (IIford Ltd., IIford, Essex). Audiographs on Singul X/RP film were developed after 28 days of exposure. The relative concentrations of radioactivity in the various tissues were estimated by visual inspection.
Limit of detection: the minimum tissue radioactivity concentration that could be detected was 4-10 dpm/mg wet tissue.

3) TLC and HPLC
Sample preparation:
Radioactivity in urine was adsorbed on Amberlite XAD-2 resin (Rohm} and Haas Co., Philadelphia, PA, USA.), washed with water and eluted with methanol. Deconjugated samples of urine (see below for procedure) were applied directly to TLC plates and analyzed by HPLC.
Aliquots of the methanol extracts of feces from rats receiving the high dose were concentrated by evaporation and then applied to TLC plates.

TLC:
Plate: pre-layered Kieselgel F25I, plates (E. Merck A.G., Darmstadt, Germany)
Developing solvents: cyclohexane : p-dioxan : acetic acid, (60 : 40 : 2, v/v)
Radioactive components were detected by either apposition autoradiography using Singul - X/RP film (Ceaverken A.B., StrangnSs, Sweden) and/or by using a Berthold Automatic TLC Linear Analyser (Models LB 283 and LB 3500 : Laboratory Impex Ltd., Twickenham, UK)

HPLC:
Instrument: Waters
AL 202 chromatograph (Waters Assoc., Northwich, U.K.) equipped with a M6000 pump, a U6K injector and a Perkin Elmer LC-55 variable wavelength UV detector.
Mobile Phase: methanol : aqueous ammonium acetate (40 : 60, v/v) adjusted to pH 6

CONJUGATION:
Samples of the eluted urine radioactivity XAD-2 columns were evaporated to dryness, reconstituted in water, adjusted to pH 5 with acetic acid and incubated with sufficient P-glucuronidase/sulphatase (Type HI, Helix pomatia, Sigma Chemical Co., Ltd., Poole, Dorset, UK).

Statistics:

No statistical analysis was performed.

Results and discussion

Preliminary studies:

The two rats (1 male, 1 female) were housed individually in glass metabolism cages and were dosed once orally with 2 mg/kg bw of the radiolabelled test material. Urine, feces and expired air were collected. The sampling intervals for urine were 0-8, 8-24, 24-48, 48-72, 72-96 and 96-120 h after administration; sample interval for feces was from 24 h to 120 h after dosing. The carbon dioxide in expired air was measured between 0-24 h and 24-48 h by trapping in an ethanolamine: 2-ethoxyethanol mixture (1:4, v/v). Five days after dosing, the animals were sacrificed by cervical dislocation and the interiors of their cages were washed with water.
The preliminary study revealed that over the period of 5 days, for males, 90.39% of the initial administered radioactivity was recovered in urine; for females it was 76.54%. In feces, the recovered radioactivity amounted from 1.43% in males and 2.54% in females. No radioactivity was recovered in the expired air.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

For both, male and female rats treated by single 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:

Tissue distribution differed between sexes. Radioactivity in plasma of females was higher than in any other tissues, followed by liver, thyroid and kidneys. In males, the highest levels of radioactivity were seen in kidneys, followed by liver and plasma.

Main ADME results 3

Type:

excretion

Results:

Excretion mainly involved urine (up to 88%) and was faster in males (i.e. 48h) as compared to females. Fecal excretion was comparatively minor (up to 8%) for both sexes.

Main ADME results 4

Type:

metabolism

Results:

The parent compound was rapidly hydrolyzed to Bromoxynil phenol, and mainly recovered in urine; parent compound only could be recovered at small amounts in feces mainly in the first 24 h.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption:
For both, male and female rats treated by single oral dosing with either 2 or 20 mg/kg bw of radiolabelled test material, absorption from the gastrointestinal tract was rapid and extensive (ca. 90% of the initially administered dose), as evidenced by the high urinary excretion and the radioactivity recovered in tissues and carcasses.

Pharmacokinetics:
In male and female rats of the low dose group (Group 3), plasma concentrations of radioactivity peaked 7 h after administration in males (11.8 µg/mL) and females (15.5 µg/mL). For males, concentrations then declined to 7.77 µg/mL at 24 hours and, with a half-life time of 43.8 hours, to 0.061 µg/L at 336 hours. For females, concentrations then declined to 8.93 µg/mL at 24 hours and thereafter with a half-life time of 57 hours, to 0.317 µg/mLat 336 hours.

In male and female rats of the high dose group (Group 4), plasma concentrations of radioactivity peaked 7 h after administration in males (102 µg/mL) and females (147 µg/mL). For males, concentrations then declined to 52.7 µg/mL at 24 hours and, with a half-life time of 36.4 hours, to 0.110 µg/L at 336 hours. For females, concentrations then declined to 127 µg/mL at 24 hours and thereafter with a half-life time of 49.7 hours, to 1.62 µg/mL at 336 hours.

For details, please refer to Attachment 1.

Details on distribution in tissues:

In Group 1 treated with 2 mg/kg bw of radiolabelled test material, the concentrations of radioactivity in the tissues of male rats sacrificed at 168 hours after single oral treatment were highest in plasma (0.257 µg/mL), as well as in liver (0.256 µg/g) and kidneys (0.270 µg/g). No radioactivity was detected in the thyroids. In females, concentrations in all tissues were significantly higher than in males. In fact, concentrations were highest in plasma (0.808 µg/mL), kidneys (0.712 µg/g) and liver (0.690 µg/g); further smaller amounts were also detected in the thyroids (0.238 µg/g), ovaries (0.190 µg/g), lungs (0.174 µg/g), heart (0.120 µg/g) and bone marrow (0.108 µg/g).

In Group 2 treated with 20 mg/kg bw of radiolabelled test material, the concentrations of radioactivity in the tissues of male rats sacrificed at 168 hours after single oral treatment showed a similar distribution to that at the lower dose level of 2 mg/kg bw. Thus, the highest concentrations were found in plasma (0.939 µg/mL), kidneys (0.907 µg/g) and liver (0.898 µg/g). With respect to the thyroids, the concentrations were significant in 2 of the 5 animals which had the higher plasma levels. In females, the mean concentration of radioactivity in plasma (11.703 µg/mL) was found to be several fold higher than that found in any other tissues; this was followed by the liver (4.614 µg/g), the thyroids (4.451 µg/g) and the kidneys (3.872 µg/g).

In Group 5 (5 females treated with 20 mg/kg bw of radiolabelled test material as single oral dose), whole-body autoradiography of the animals sacrificed at various times was in line with the results of the quantitative tissue distribution reported above. In fact, highest levels of radioactivity were observed at 7 hours after dosing when relatively high levels were associated with the blood, liver, skin, epimysia and lung. Radioactivity was widely distributed throughout other tissues, with trace levels occurring in the brain and spinal cord. Radioactivity concentrations declined relatively slowly with time and at 14 days after dosing, trace levels were still observed in several tissues.

For further details, please refer to Attachment 2.

Transfer into organs

Key result

Transfer type:

other: transfer from plasma into organs

Observation:

other: indicated by plasmacokinetics

Details on excretion:

With respect to the male rats treated with the low dose of 2 mg/kg bw (Group 1), means of 87.54% and 3.63% of initial administered dose were excreted in the urine and feces respectively during 7 days. The rate of urinary excretion in male rats decreased with a half-life of 28.3 hours. At sacrifice, a mean of 2.69% dose was retained in the animal of which 0.85% was found in the skin and fur. In female rats of this group, means of 80.17% and 5.35% were excreted in the urine and feces respectively during 7 days. Urinary excretion decreased with a half-life of 42.7 hours and at sacrifice, a mean of 6.28% dose was retained in the animal of which 1.84% was found in the skin and fur.
Since, the preliminary experiments showed no radioactivity was eliminated in the expired air, this was not further considered.

With respect to the male rats treated with the high dose of 20 mg/kg bw (Group 2), means of 87.44% and 3.46% of initial administered dose were excreted in the urine and feces respectively during 7 days. The rate of urinary excretion was similar to that obtained at the low dose level with a half-life of 23.4 hours. After 7 days, male rats retained 1.27% dose of which 0.52% was in the skin and fur and 0.46% dose in the carcass. In the female rats of this group, means of 76.29% and 7.12% dose were excreted in the urine and feces, respectively. The rate of urinary excretion was similar to that obtained at the low dose level with a half-life of 50.3 hours. In females, a mean of 8.32% dose was retained of which 2.60% was in the skin and fur and 4.28% was in the carcass.

For further details, please refer to Attachment 3.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

Cmax: low dose: 11.8 µg equiv/mL (males), 15.5 µg equiv/mL (females); high dose: 102 µg equiv/mL (males), 147 µg equiv/mL (females);

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

Tmax: 7 h

Toxicokinetic parameters 3

Key result

Toxicokinetic parameters:

half-life 1st: males: 36.4 - 43.8 h, females: 49.7 - 57.0 h (in blood)

Toxicokinetic parameters 4

Key result

Toxicokinetic parameters:

half-life 1st: males: 23.4 - 28.3 h, females 42.7 - 50.3 h (urinary excretion)

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

In both male and female rats, a single major metabolite was identified in urine as Bromoxynil. In the 0-8, 8-24 and 24-48-h samples, some readily hydrolysed conjugates of Bromoxynil phenol were present, which accounted for 15% and 10.6% dose in male and female rat urine respectively. Free Bromoxynil phenol accounted for 65.7% and 58.5% dose in the 0-168-h urine of male and female rats respectively. The parent compound was not be detected in any of the urine samples.
With respect to the feces, two major components were identified, one of them being the metabolite Bromoxynil phenol whereas the second component referred to the parent compound, i.e., unchanged test material.
For further details, please refer to Attachment 4.
For metabolic pathway, please refer to Attachment 5.

Enzymatic activity

Enzymatic activity measured:

No

Applicant's summary and conclusion

Conclusions:

The toxicokinetic behavior and metabolism of the test compound was investigated in a GLP-compliant study similar to OECD 417 (1984). During the study, rats of both sexes were orally treated with a single dose of either 2 or 20 mg/kg bw of the radiolabelled test material. The study is considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat.
The test substance was well absorbed from the gastrointestinal tract.
In male and female rats treated with the lower dose, plasma concentrations of radioactivity peaked 7 h after administration in males and then declined over time, with a half-life time of 43.8 hours for males and 57 hours for females. With respect to the high dose level, plasma concentrations of radioactivity also peaked 7 h after administration for both, males and females. Thereafter the concentration declined over the course of time with a half-life of 36.4 h for male and 49.7 h for females.
Following treatment with the lower dose (2 mg/kg bw of radiolabelled test material), the concentrations of radioactivity in the tissues of male rats sacrificed at 168 hours were highest in plasma (0.257 µg/mL), liver (0.256 µg/g) and kidneys (0.270 µg/g). No radioactivity was detected in the thyroids. In females, concentrations in all tissues were significantly higher than in males. In fact, concentrations were highest in plasma (0.808 µg/mL), kidneys (0.712 µg/g) and liver (0.690 µg/g); further smaller amounts were also detected in thyroids (0.238 µg/g), ovaries (0.190 µg/g), lungs (0.174 µg/g), heart (0.120 µg/g) and bone marrow (0.108 µg/g). Similar distributions were noticed following single oral administration of the higher dose (20 mg/kg bw of radiolabelled test material). For males, the highest concentrations were found in plasma (0.939 µg/mL), kidneys (0.907 µg/g) and liver (0.898 µg/g). In females, the mean concentration of radioactivity in plasma (11.703 µg/mL) was found to be several fold higher than that found in any other tissues. This was followed by the liver (4.614 µg/g), the thyroids (4.451 µg/g) and the kidneys (3.872 µg/g). Whole-body autoradiography of the animals sacrificed at various times was in line with the results of the quantitative tissue distribution reported above.
The main route of excretion was the urine, which accounted for approximately 87% for males and 78% for females of the initially administered dose; fecal excretion was minor. The metabolism of the test substance was not sex dependent. Bromoxynil was identified as major metabolite in urine, which resulted from the hydrolysis of the octanoyl group of the parent compound. Parent compound was not identified in urine samples, but to a small amount in feces.