2,6-dibromo-4-cyanophenyl octanoate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

absorption

distribution

excretion

metabolism

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

Radiolabelling:

yes

Remarks:

14C ring radiolabel

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

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

Dose / conc.:

2 mg/kg bw (total dose)

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.

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.

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.

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.

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.

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.

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.

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.

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);

Key result

Toxicokinetic parameters:

Tmax: 7 h

Key result

Toxicokinetic parameters:

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

Key result

Toxicokinetic parameters:

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

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 measured:

No

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.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Sep 1992 - 29 Jun 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C ring radiolabel

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 7 - 11 weeks
- Weight at study initiation: 251 - 277 g (males) 206 - 226 g (females)
- Housing: up to 5 per cage according to sex, in wire floor polypropylene cages suspended over polypropylene dirt trays containing soft white wood sawdust (Special Diet Services, Stepfield, Witham, Essex)
- Diet: commercial pellet diet, SQC Rat and Mouse Maintenance Diet No. 1, Expanded (Special Diet Services, Stepfield, Witham, Essex), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: approximately 1 week
- Health status: all animals were examined for signs of ill-health
- Fasting period: overnight before and 3 h after administration of the radiolabelled test article

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 22 Oct To: 23 Jun 1993

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test article was prepared as a suspension in polyethylene glycol 400 to provide a nominal dose volume of 5 mL/kg body weight. The non-radiolabelled formulation was prepared in two batches, one for Days 1 to 7 and one for Days 8 to 14. The stock formulations were kept at 0 to 4°C throughout the dosing period and subsamples were removed daily as required immediately prior to each dosing occasion. The radiolabelled formulation was prepared as one batch on the day of dosing.

Duration and frequency of treatment / exposure:

Oral repeated dose experiment over 15 days

Dose / conc.:

2 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

5

Control animals:

no

Positive control reference chemical:

no

Details on study design:

The repeated dose group was administered non-radiolabelled test substance once daily for 14 days, followed by a final dosing with radiolabelled test material on day 15.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, feces, cage washes, tissues (brain, blood, abdominal fat, gonads, heart, kidney, muscle, liver, spleen, thyroid, uterus, skin, gastrointestinal tract plus contents, lungs, bone, bone marrow, residual carcass and gross lesions)
- Time and frequency of sampling:
Urine: 0 - 8, 8 - 24, and in 24 h intervals thereafter, until 168 h post-dose.
Feces: in 24 h intervals after dosing until 168 h post-dose.
Cage wash: after each excreta collection, the cage was rinsed with water and the water collected.
- Tissues: at 168 h post-dose, animals were exsanguinated under halothane anesthesia and the tissues (including blood) were collected.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, feces, cage washes
- Time and frequency of sampling: for urine: 0 - 8, 8 - 24, and in 24 h intervals thereafter, until 168 h post-dose; for feces: in 24 h intervals after dosing until 168 h post-dose.
- From how many animals: samples containing the highest radioactivity were pooled by sex and time ranges and were collected for urine in 0 - 8, 8 - 24, 24 - 48 and 48 - 72 h intervals for males, and in 0 - 8, 8 - 24, 24 - 48, 48 - 72 and 72 - 96 h intervals for females; feces were collected in 24 h intervals until 48 h (males and females).

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 separation and evaluation of compounds, high performance liquid chromatography (HPLC) and/or thin layer chromatography (TLC) with radio detection were used.
3) For identification of compounds mass spectroscopy was used (GC-MS)

1) LSC:
The following liquid scintillation counters used:
Beckman (Beckman Instruments, High Wycombe, Bucks.) or Packard Tri-Carb liquid scintillation counters (Canberra Packard, Pangbourne, Berks.)

Sample preparation:
Urine and cage washings were added directly to scintillant and assayed by liquid scintillation counting.
Feces and cage debris were homogenized in methanol and centrifuged. The separated residue was air-dried and combusted. The volumes of the fecal organic extracts were recorded and aliquots submitted to liquid scintillation counting. Bone marrow was added directly to ashless floc and combusted. Bone was freeze-dried, crushed and ground using a pestle and mortar and combusted. Combustion was performed in oxygen using a Packard Sample Oxidiser. The combusted products were absorbed in Carbo-Sorb, mixed with Permafluor V, and the radioactivity determined by liquid scintillation counting.
Liver and gastrointestinal tract (plus contents) were homogenized in deionised water.
Ovaries and thyroids were analyzed as whole tissues.
All other tissues were macerated with scissors.
The residual carcass was solubilized using 40% (w/v) potassium hydroxide in methanol.

Solubilization was performed by adding Soluene-350 solubilizing agent (CanberraPackard, Pangbourne, Berkshire) to blood and tissue samples and adding liquid scintillant after incubation.

2) HPLC and TLC

Urine samples and fecal extracts were analyzed by HPLC and TLC (urine only) with radio detection. Therefore, metabolites from the fecal samples were extracted with methanol. For urine, no extraction was done but they were passed through Amberlite columns (20 x 1.5 cm) containing XAD-2 resin. Aliquots of reconstituted urine extract were added to glass vials and 0.2 M ammonium acetate buffer, (pH 5) and helix pomatia extract (400 µL) admixed. Incubation lasted for 18 h in a water bath at 37 °C.

HPLC System:
Column: Spherisorb S5 ODS S2
Mobile phase: (A) 0.05M ammonium acetate pH 6 and (B) methanol

TLC: methanol urine concentrates were spotted onto TLC plates with standards, these were developed in the solvent system. The plates were then air-dried and viewed under UV (254 nm) prior to analysis on a RITA linear analyzer.
Plate: Silica gel 60 F254
Solvent system: cyclohexane:p-dioxan:acetic acid (60:40:2 v/v/v)

3) GC-MS
Samples tested: urine samples (concentrated methanol extracts) 24 h(males) and 48 h (females). Feces could not be analyzed because the level of radioactivity was too low.

System: Varian 3400 Gas chromatograph, Finnigan MAT 8200 magnetic sector double focusing mass spectrometer
Column: CP SIL 8CB
To aid identification, reference articles were similarly processed and analyzed. Data were collected using scan mode.

- Limits of detection: The limit of detection for the analysis of each sample type was taken as twice the background disintegration rate obtained from the measurement of blank samples of the same type.

Statistics:

Not performed.

Preliminary studies:

None

Type:

absorption

Results:

For both, male and female rats treated by 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.

Type:

distribution

Results:

In females, radioactivity blood levels in females were 7-fold higher than in males; radioactivity was detected in all female tissues at 168 h, with highest levels found in liver, kidney and thyroid.

Type:

distribution

Results:

In males, radioactivity levels were below detection limit in many of the tissues assayed, and was not detectable at all in the thyroid.

Type:

excretion

Results:

Excretion mainly involved urine (80.2% in males; 67.9% in females) and was faster in males (i.e. 48 h) as compared to females. Fecal excretion was minor (7% in males, 9.9% in females).

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.

Details on absorption:

Recovery:
Over 96% of the administered radioactivity was recovered for both males and females.

Absorption:
For both, male and female rats treated by repeated dosing, the test material was rapidly and extensively absorbed from the gastrointestinal tract (> 85% of initial dose), as evidenced by the high excretion level mainly involving urine (> 65% radioactivity recovery, and additionally, 9.1% for males and 10.7% for females obtained from cage washes, also attributed to urine), and to a lesser extent, the feces (ca. 6 .9 to 9.9%).

For details, please refer to Attachment 1.

Details on distribution in tissues:

The distribution of radioactivity in the carcass and tissues showed sex differences, with more widespread distribution and higher levels of radioactivity found in the tissues and carcasses of female animals, as compared to males. In fact, tissue radioactivity levels in males were ca 4 times lower than those found in females (0.61% vs 2.67%). For males, the organs with the highest radioactivity were liver (0.23 µg equiv/g) and kidney (0.24 µg equiv/g), while there was no radioactivity found in the brain or thyroid. In females, the thyroid contained the highest concentration of radioactivity (1.12 µg equiv/g), followed by liver (0.80 µg equiv/g), blood (0.70 µg equiv/g), kidney (0.56 µg equiv/g), uterus (0.44 µg equiv/g) and ovaries (0.39 µg equiv/g). In all other tissues of female rats, radioactivity concentrations equal to or less than 0.350 µg equiv/g were detected.
While males excreted more of the radioactivity via the urine, females retained a greater proportion of the dose in the carcass (males, 0.872%; females 5.363%).

For details, please refer to Attachment 3.

Key result

Test no.:

#1

Transfer type:

other: transfer from plasma into organs assumed

Observation:

not determined

Details on excretion:

Excretion mainly involved the urine (80.2% of the administered dose in males; 67.9% in females); fecal excretion was comparatively low, amounting for 6.9% of the administered dose in males and 9.9% in females.
In fact, excretion of via urine was greatest in males over the first 48 h, with 25.8% and 22.6% eliminated at 24 h and 48 h post-dose. After the 72 h sampling interval, less than 4% of the dose was recovered at each sampling interval. For females, renal elimination was comparatively reduced and prolonged in time. Recovery of radioactivity within 48 h accounted for 31.5% for females compared with 62.7% for males. However, at later times a larger proportion of the dose was present in female urine with 2.8% being found at the last sampling interval (144 to 168 h). The delayed elimination of radioactivity via the urine for females was also reflected by the mean urinary excretion rate after 8 h, which was 2.5 µg/h compared to 11.7 µg/h in males.
Radioactivity in feces was recovered mainly during the first 48 h after dosing.

In the cage washes, 9.1% and 10.7% of the initially administered dose were recovered for males and females, respectively. This was considered to be associated with the renal elimination, and thus, additioned to the factual urine excretion.

For details, please refer to Attachment 2.

Metabolites identified:

yes

Details on metabolites:

Male urine
The main metabolite (T2) identified via TLC resulted from the hydrolyze of the parent compound and was identified as Bromoxynil phenol. Via HPLC, Bromoxynil phenol was also identified in urine (as U2), accounting for 14.24% of the initial radioactivity dose level. Two further radioactive fractions were detected prior to deconjugation (U1 and U2, accounting for 0.49 - 0.70% and 9.05 - 25.5% of the dose, respectively). After deconjugation, U5 was identified as a minor component in the 0 - 8 h urine extract (0.59% of the dose). At all other times (8 to 24, 24 to 48 and 48 to 72 h) after deconjugation, U1 was not detected even in the presence of a glucuronidase inhibitor and the radioactive fraction (U2) had increased proportionately. This component accounted for > 99% of the dose in each urine pool. The parent compound was not detected in the urine.

Male feces
Due to low radioactivity in feces, only that from 0 - 24 and 24 - 48 h collection intervals were profiled by HPLC. Three radioactive fractions were found, two in both samples, a third minor one only in the 24 h-interval (FETX2). In the 0-24 h interval, the parent compound was the major compound found in feces (FEXT3, 1.36% of the initial dose). FEXT1 was identical with U2 and T2 and thus was identified as Bromoxynil phenol; it accounted for 0.32% of the dose at the 0 - 24 h sampling interval. After 48 h, the parent compound was present at relatively low levels (0.09%), while Bromoxynil phenol was the major component (0.88% of the initial dose) in this interval.

Female urine
The main metabolite identified via TLC resulted from the hydrolyze of the parent compound and was identified as Bromoxynil phenol (T2). Via HPLC, an additional fraction (U4) was found (0 - 8 h and 24 - 48 h urine representing 0.18% and 1.31% of the dose in each pool respectively). At the 8 h time point, Bromoxynil phenol (U2) was identified in the urine (4.13% of the initial dose). At all other time points, two radioactive compounds were found: U1 representing 1.06 to 2.84% of the intial dose while Bromoxynil phenol (U2) represented the main metabolite, accounting for 10.97 to 20.52% of the initial dose.
After deconjugation, all urine extracts contained Bromoxynil phenol (U2). This component represented almost all of the recovered radioactivity in urine and only the 24 to 48 h urine pool showed the presence of a second region (U4) corresponding to 1.38% of the initial dose; U4 was not seen in the urine of males.


Female feces
The radioactive components present in females feces were similar to those of males, but they differed in quantity and proportion. Bromoxynil phenol (i.e., FETX1, (identical to U2 and T2) was the dominant component at both 24 and 48 h representing 0.97% and 0.89% of the initially administered dose of radioactivity, respectively. The parent compound (FEXT3) was also detected in feces (0.58% after 24 h and 0.12% after 48 h).

For further details, please refer to Attachment 4.
The proposed metabolic pathway is available from Attachment 5.

Enzymatic activity measured:

None

Bioaccessibility (or Bioavailability) testing results:

None

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, male and female rats were orally treated for 14 days with 2 mg/kg bw/day of the unlabelled test substance once a day, followed by a single oral administration of 2 mg/kg bw/day of 14C-labelled 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.
Excretion mainly involved the urine, with radioactivity recovery amounting for approximately 80% of the initially administered dose in males and 68% in females. Fecal excretion was minor. Sex differences in the urinary excretion rate resulted in there being a more widespread distribution and higher levels of radioactivity present in the tissues and carcasses of female animals, as compared to males. Radioactivity was almost distributed in liver and kidney. The metabolism of the test substance was not sex-dependent and basically, involved hydrolysis of the octanoyl group of the parent compound, resulting in Bromoxynil as major metabolite. Parent compound was not identified in urine samples, but to a small amount in feces.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Apr 1984 - 11 Jan 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

adopted 2004

Deviations:

yes

Remarks:

i.a., small group size and thus, no interim sacrifice at end of exposure, acclimation period belong guideline recommendation, exposure period above guideline recommendation

Principles of method if other than guideline:

No guideline mentioned in the study report, however, the test conduct is in principle compliant to the OECD test guideline 427.

GLP compliance:

yes

Remarks:

with following deviations: No standard operating procedure available for receiving rodents In the Radiation Building during the course of this study; Dosing solutions not analyzed for for purity and stability of the test chemical.

Radiolabelling:

yes

Remarks:

14C ring radiolabel

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale, Pennsylvania, USA
- Age at study initiation: 7 - 9 weeks
- Weight at study initiation: 265 - 286 g
- Fasting period before study: no
- Housing: during acclimation, rats were housed in groups of three in shoe-box cages with Ab-sorb-dri bedding (Garfield, New-Jersey), after application, rats were housed individually.
- Individual metabolism cages: Roth-type metabolism cages designed for the separate collection of urine, feces and expired gases.
- Diet: basic diet of Agway, Pro-Lab, certified pelleted rodent chow (RMH 3000; Agway Inc., St. Marys, Ohio), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 4 days

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

IN-LIFE DATES: From: 2 To: 6 Apr 1984

Type of coverage:

occlusive

Vehicle:

acetone

Remarks:

only for the applied dose of 1037 µg/25 cm2 corresponding to 46.9 µCi; for the remaining doses, water was used as vehicle.

Duration of exposure:

48 h

Doses:

67, 137 and 1037 µg/25 cm2, equivalent to 3, 6.2 and 46.9 µCi, respectively

No. of animals per group:

3 µCi:1 animal
6.2 µCi : 1 animal
46.9 µCi: 2 animals

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The test substance radiolabelled with 14C was received in 4.2 mL toluene. Two 1 mL aliquots of the toluene solution were evaporated to dryness. One was dissolved in 2.6 mL acetone and the other suspended In 2.6 mL water. The water solution was sonicated before dosing.

APPLICATION OF DOSE:
For the rats receiving the highest dose of radiolabelled test material in acetone, the dose was applied to the 25 cm2 skin application area in two 0.5 mL aliquots. For the rats receiving the lower doses of radiolabelled test material in water, each dose was applied in 3 to 4 aliquots.

VEHICLE
- Justification for use and choice of vehicle: chosen as appropriate
- Amounts applied: 1 mL

TEST SITE
- Preparation of test site: one day before the experiment, the hair at the test side was clipped, a neoprene rubber template was glued in place and rats were surgically implanted with an indwelling cannula in the right external jugular vein under Metofane anesthesia (for blood withdrawal).
- Area of exposure: 25 cm²
- Type of cover / wrap if used: polyethylene wrap
- Time intervals for shavings or clippings: on the day before the experiment

SITE PROTECTION:
To prevent oral ingestion, the polyethylene wrap used to cover the application area, was secured with Elastikon tape.

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: 1:1 mixture of acetone and water
- Time after start of exposure: 48 h
The washings and polyethylene wrap were stirred continuously for approximately 6 h in 1 L of methanol and 1 mL samples were removed for radiometrlc analyses.

SAMPLE COLLECTION
- Collection of blood: yes, 0.1 - 0.2 mL, 30 min and 1, 2, 4, 8, 12, 24, 32 and 48 h following dosing and 30 min and 1, 2, 4, 8, 12, 24, 32 and 48 h post-washing
- Collection of urine and feces: yes, daily during exposure and daily for 48 h post-washing.
- Collection of expired air: yes, expired volatiles were trapped in acetonitrile and collected at 8, 24 and 48 hours post-application and at 8 h post-washing. Expired CO2 was collected in ethanolamine/2-methoxyethanol traps (3:7 v/v) at 8, 24 and 48 hours post-dose application and following the same schedule following the skin wash.
- Cage wash: yes, each cage was washed daily with a 1:1 mixture of acetone and water.
- Terminal procedure: 96 h post-application, rats were anesthetized with Metofane and exsanguinated via the abdominal aorta
- Analysis of organs: skin from the dosing area (solubilized in 200 mL of 10N NaOH), remaining carcass (solubilized in 2 L of 10N NaOH).

SAMPLE PREPARATION
- Storage procedure: The stock sample was stored at ca. 4 °C.
- Preparation details: Samples of plasma, blood cells, cage and skin washings, acetonitrile (trapping solution) and urine were counted directly in Aquasol-II. Aliquots (1 mL) of the trapping solution from the C02 traps were analyzed using a dioxane-based scintillator. Triplicate samples of NaOH solubillzation mixtures were diluted 10-fold with deionized water and aliquots (1 mL) of these dilutions were counted in Dimilume-30.

ANALYSIS
- Method types for identification: Liquid scintillation counting (LSC)
- Validation of analytical procedure: Standards for radioactivity determination were prepared by diluting weighed 0.1 mL samples of each dosing solution 100 fold in methanol. 0.1 mL of this dilution was counted according to standard operating procedures. The measured amounts of radioactivity in the dosing solutions were 46.9 µci (1037 µg) in the acetone vehicle and 6.2 µci (137 µg) and 3.0 µci (67 µg) in the two water vehicle suspensions. The amounts of radioactivity differed between solvents because of unequal solubility of the radiolabelled chemical.
- Limits of detection and quantification: not reported

The pharmacokinetic behavior of the test substance was evaluated using the BASIC computer program, ESTRIP. Terminal half-life (t 1/2) was calculated as follows:

t1/2 (h) = (0.693)/k

k = rate constant for terminal exponential phase

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

All percentages of recovery reported below are over the total exposure and recovery period, for details, please refer to Attachment 1.

- Skin (washed): 6.61 and 6.95% (acetone vehicle), 10.46 and 8.02% (water vehicle)
- Carcass: 8.41 and 5.31% (acetone vehicle), 6.07 and 13.46% (water vehicle)
- Urine: 17.14 and 23.7% (acetone vehicle), 17.19 and 9.65% (water vehicle)
- Cage wash + cage wipe: 4.44 and 3.95% (acetone vehicle), 5.09 and 6.51% (water vehicle)
- Feces: 3.38 and 5.92% (acetone vehicle), 2.7 and 7.14% (water vehicle)
- Expired air: < 0.2%
- Dosing rod and template: 4.76 and 2.2 % (acetone vehicle), 5.4 and 4.62% (water vehicle)
- Skin washings and polyethylene wrap: 46.4 and 40.42% (acetone vehicle), 51.43 and 46.05% (water vehicle)

- total percentage of radioactivity recovered from wrapping, washing fluid, dosing rod and template: 42.62 and 51.16% (acetone vehicle), 50.67 and 56.83% (water vehicle)
- total percentage of radioactivity recovered in urine, feces, expired air, carcass and skin: 40.01 and 45.86% (acetone vehicle), 41.61 and 45.07% (water vehicle)

- Blood: For blood, a pharmacokinetic investigation was conducted. The nanogram equivalents of the radiolabeled test substance were measured in plasma during exposure and up to 48 h after the end of administration. During exposure, the radioactivity values for the test substance applied in acetone were maximal for the 12 to 24 h period (3781.0 ng/g) in one animal and 24 to 32 h for the other animal (3836.6 ng/g) and declined for subsequent samples. With the water
vehicle, the radioactivity in plasma during exposure was at a maximum for the 24 to 32 h sample with one animal and at a plateau during the 12 to 24 h and 24 to 32 h sample periods with the second animal. Radioactivity in the plasma after washing the dosing solution from the skin decreased slowly and similarly for all animals dosed with the radiolabelled test substance. Radioactivity in red blood cells was negligible and was not tabulated.

For further details, please refer to Attachment 2.

Total recovery:

- Total recovery: The total recovery of radiochemical was similar for both vehicles with values of 88.48 and 91.17% of the applied dose for acetone and 95.74 and 98.44% of the applied dose for water.
- Recovery of applied dose acceptable: yes

Key result

Time point:

48 h

Dose:

46.9 μci (acetone as vehicle)

Parameter:

percentage

Absorption:

>= 40 - <= 46 %

Remarks on result:

other: i.e., percentage of administered dose based on radioactivity recovery in animal tissues and excreta

Key result

Time point:

48 h

Dose:

3 and 6.2 μci (water as vehicle)

Parameter:

percentage

Absorption:

>= 41 - <= 45 %

Remarks on result:

other: i.e., percentage of administered dose based on radioactivity recovery in animal tissues and excreta

Conversion factor human vs. animal skin:

No data reported.

Radioactivity recovered from the application materials and in the fluid used to wash the skin was in the range of 42.5 to 51.5% of the administered dose of 46.9 µCi in acetone; radioactivity recovered from the application materials and in the fluid used to wash the skin was between 50 and 57% of the administered doses in water (3 and 6.2 µCi).

The half-life (t 1/2) In hours for the post-washing (elimination) phase was reported as 40.8 and 27.7 hours for the dose in acetone as vehicle and as 25.7 and 30.1 hours for the doses in water as vehicle.

For further details, please refer to Attachment 3.

Conclusions:

The dermal absorption of the test compound was investigated in a GLP-compliant study conducted similar to OECD 427. During the study, males rats received single dermal applications of the radiolabelled test material at doses of 1037 µg/25 cm2 of skin (acetone used as vehicle, 2 rats), 137 µg/25 cm2 of skin (water as vehicle, one rat)and 67 µg/25 cm2 of skin (water as vehicle, one rat) for 48 h. These doses corresponded to 46.9, 6.2 and 3 µCi, respectively. The application volume for each dosage was 1 mL. Treatment was followed by a recovery period and the animals were finally sacrificed after 96 h post-treatment. The study is considered valid, scientifically acceptable and appropriate for the assessment of dermal absorption in the rat, despite of the low number of animals used.
The total recovery of radioactivity relative to the administered dose was 88.5% and 91.2% for the highest dose (46.9 µCi in acetone), and 95.7% and 98.4% for the lower doses (6.2 and 3 µCi in water). Thus, the choice of the vehicle had no impact on the dermal absorption. In fact, for both vehicles, the predominant route of radioactivity excretion was via the urine and the greatest relative recovery of radioactivity in the body was in the carcass and washed skin. Dermal absorption was calculated and reported as percentage of the administered dose based on recovery of radioactivity in the animal tissues and excreta. The percentage of absorbed radiolabel relative to the administered dose, over the 48-hour treatment period, was about 40 to 46% for the high dose of 46.9 µCi in acetone. The percentage of absorbed radiolabel relative to the administered dose, over the 48-hour treatment period, was about 41 to 45% for the lower doses in water (i.e., 6.2 and 3 µCi).
Radioactivity recovered from the application materials and in the fluid used to wash the skin was about 42 to 51.5% of the administered dose for 46.9 µCi in acetone, and 50.5 to 57% for the lower doses in water (i.e., 6.2 and 3 µCi).
The half-life (t 1/2) In hours for the post-washing (elimination) phase was reported to be 40.8 and 27.7 hours for the high dose of 46.9 µCi in acetone, and 25.7 and 30.1 hours for the doses in water (i.e., 6.2 and 3 µCi).

Description of key information

Absorption of the test substance is to be taken into consideration after oral and dermal exposure and inhalation. However, dermal exposure is considered to be rather low, with 40 – 46% absorption. Acute oral exposure and inhalation led to systemic toxicity and mortality, while no systemic effects were observed after acute dermal exposure.

With respect to the oral route, biokinetic studies showed that the test substance was absorbed to a high degree from the intestinal lumen. Following absorption, the test substance was transported by the blood and distributed to different organs and tissues, especially the liver and kidney. The metabolization rate of the test substance in rodents was high and Bromoxynil phenol was identified as the main metabolite. Elimination of ca. 80% of the test substance happened within the first 72 hours by males and 96 hours by females. Elimination primarily occurred via the urine, while < 10% was excreted via the feces. There is no indication of any bioaccumulation potential of the parent compound and/or its metabolites.

Key value for chemical safety assessment

Absorption rate - oral (%):

85

Absorption rate - dermal (%):

46

Additional information

Three studies are available, providing reliable informations of absorption, distribution, excretion and metabolization of the test substance in rodents; all three studies are considered suitable to be used as key studies. In fact, of the three studies, two deals with basic toxicokinetics, metabolism, distribution and excretion (M-162043-01-1 and M-262277-01-1) whereas the third one refers to a dermal absorption study (M-283357-01-1).

The toxicokinetic and metabolic behavior of the test substance was investigated in the rat in a GLP compliant study according according to EPA OPP 85-1 (M-162043-01-1, 1993). During the study, 5 animals per sex were administered the non-radiolabelled test substance for 14 days (2 mg/kg bw/day). On the 15th day, all rats received 2 mg/kg bw of the radiolabelled test substance. Urine, feces and cage wash were collected. Exhaled carbon dioxide was not trapped during this study as previous studies with the test substance had shown that the expired air was not a route of elimination (M-262277-01-1). At sacrifice, carcass, blood, organs and tissues were sampled. Radioactivity of the samples was analyzed by liquid scintillation counting (LSC). Urine, fecal and liver samples were further analyzed by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC, urine only) with radiodetection for metabolite identification.

Over 96% of the administered radioactivity was recovered for both males and females rats. For both, male and female rats treated by repeated dosing, the test material was rapidly and well absorbed from the gastrointestinal tract (> 85% of initial dose). This was evidenced by the high excretion level mainly involving urine (> 65% radioactivity recovery in urine, as well as additional 9.1% for males and 10.7% for females obtained from cage washes, which also were attributed to urine). Excretion via feces was minor (ca. 6.9 to 9.9% of initial dose).

The distribution of radioactivity in the carcass and tissues showed sex differences, with more wides pread distribution and higher levels of radioactivity found in the tissues and carcasses of female animals, as compared to males. In fact, tissue radioactivity levels in males were ca 4 times lower than those found in females (0.61% vs 2.67%). For males, the organs with the highest radioactivity were liver (0.23μg equiv/g) and kidney (0.24μg equiv/g), while there was no radioactivity found in the brain or thyroid. In females, the thyroid contained the highest concentration of radioactivity (1.12μg equiv/g), followed by liver (0.80μg equiv/g), blood (0.70μg equiv/g), kidney (0.56μg equiv/g), uterus (0.44μg equiv/g) and ovaries (0.39μg equiv/g). In all other tissues of female rats, radioactivity concentrations equal to or less than 0.350μg equiv/g were detected.While males excreted more of the radioactivity via the urine, females retained a greater proportion of the dose in the carcass (males, 0.872%; females 5.363%).

Excretion mainly involved the urine (80.2% of the administered dose in males; 67.9% in females); fec al excretion was comparatively low, amounting for 6.9% of the administered dose in males and 9.9% in females. In fact, excretion of via urine was greatest in males over the first 48 h, with 25.8% and 22.6% eliminated at 24 h and 48 h post-dose. After the 72 h sampling interval, less than 4% of the dose was recovered at each sampling interval. For females, renal elimination was comparatively reduced, and prolonged in time. Recovery of radioactivity within 48 h accounted for 31.5% for females compared with 62.7% for males. However, at later times a larger proportion of the dose was present in female urine with 2.8% being found at the last sampling interval (144 to 168 h). The delayed elimination of radioactivity via the urine for females was also reflected by the mean urinary excretion rate after 8 h, which was 2.5μg/h compared to 11.7μg/h in males. Radioactivity in feces was recovered mainly during the first 48 h after dosing. In the cage washes, 9.1% and 10.7% of the initially administered dose were recovered for males and females, respectively. This was considered to be associated with the renal elimination, and thus, additioned to the factual urine excretion. 

Metabolites were identified in urine and feces. In males, the main metabolite (T2) identified via TLC resulted from the hydrolyze of the parent compound and was identified as Bromoxynil phenol. Via HPLC, Bromoxynil phenol was also identified in urine (as U2), accounting for 14.24% of the initial radioactivity dose level. Two further radioactive fractions were detected prior to deconjugation (U1 and U2, accounting for 0.49 - 0.70% and 9.05 - 25.5% of the dose, respectively). After deconjugation, U5 was identified as a minor component in the 0 - 8 h urine extract (0.59% of the dose). At all other times (8 to 24, 24 to 48 and 48 to 72 h) after deconjugation, U1 was not detected even in the presence of a glucuronidase inhibitor and the radioactive fraction (U2) had increased proportionately.This component accounted for > 99% of the dose in each urine pool.The parent compound was not detected in the urine. With respect to the feces, due to low radioactivity, only that from 0 - 24 and 24 - 48 h collection intervals were profiled by HPLC. Three radioactive fractions were found, two in both samples, a third minor one only in the 24 h-interval (FETX2). In the 0-24 h interval, the parent compound was the major compound found in feces (FEXT3, 1.36% of the initial dose). FEXT1 was identical with U2 and T2 and thus was identified as Bromoxynil phenol; it accounted for 0.32% of the dose at the 0 - 24 h sampling interval. After 48 h, the parent compound was present at relatively low levels (0.09%), while Bromoxynil phenol was the major component (0.88% of the initial dose) in this interval.

In females, the main metabolite identified via TLC resulted from the hydrolyze of the parent compound and was identified as Bromoxynil phenol (T2). Via HPLC, an additional fraction (U4) was found (0 - 8 h and 24 – 48 h urine representing 0.18% and 1.31% of the dose in each pool respectively). At the 8 h time point, Bromoxynil phenol (U2) was identified in the urine (4.13% of the initial dose). At all other time points, two radioactive compounds were found: U1 representing 1.06 to 2.84% of the intial dose while Bromoxynil phenol (U2) represented the main metabolite, accounting for 10.97 to 20.52% of the initial dose. After deconjugation, all urine extracts contained Bromoxynil phenol (U2). This component represented almost all of the recovered radioactivity in urine and only the 24 to 48 h urine pool showed the presence of a second region (U4) corresponding to 1.38% of the initial dose; U4 was not seen in the urine of males. With respect to the feces, the radioactive components present in feces from females were similar to those of males, but they differed in quantity and proportion. Bromoxynil phenol (i.e., FETX1, (identical to U2 and T2) was the dominant component at both 24 and 48 h representing 0.97% and 0.89% of the initially administered dose of radioactivity, respectively. The parent compound (FEXT3) was also detected in feces (0.58% after 24 h and 0.12% after 48 h).

Thus, it was concluded that under the test conditions used, thetest substance was well absorbed from the gastrointestinal tract. Excretion mainly involved the urine, with radioactivity recovery amounting for approximately 80% of the initially administered dose in males and 68% in females. Fecal excretion was minor. Sex differences in the urinary excretion rate resulted in there being a more widespread distribution and higher levels of radioactivity present in the tissues and carcasses of female animals, as compared to males. Radioactivity was almost distributed in liver and kidney. The metabolism of the test substance was not sex-dependent and basically, involved hydrolysis of the octanoyl group of the parent compound, resulting in Bromoxynil phenol as major metabolite. Parent compound was not identified in urine samples, but to a small amount in feces.

The toxicokinetic and metabolic behavior of the test substance was further investigated in the rat in a GLP compliant study for which no information on guideline was given in the report (M-162043-01-1, 1993). However, the test conduct is widely compliant to the OECD test guideline 417 as adopted 1984. 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.

Urine, feces, carcass, blood, organs and tissues were collected for analysis. Radioactivity in the samples was assessed by means of by liquid scintillation counting (LSC). Urine and fecal samples were further analyzed by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC, urine only) with radiodetection for metabolite identification. Expired air was not sampled since a preliminary study with 1 male and 1 female rat showed, that no radioactivity was eliminated in the expired air. 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.

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. Regarding pharmacokinetics, in male and female rats of the low dose group (Group 3), plasma concentrations of radioactivity peaked 7 h after administration in males (11.8μg/mL) and females (15.5μg/mL). For males, concentrations then declined to 7.77μg/mL at 24 hours and, with a half-life time of 43.8 hours, to 0.061μg/L at 336 hours. For females, concentrations then declined to 8.93μg/mL at 24 hours and thereafter with a half-life time of 57 hours, to 0.317μg/mL at 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 127μg/mL at 24 hours and thereafter with a half-life of 49.7 hours, to 1.62μg/mL at 336 hours. With respect to distribution, 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 obs erved 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. Regarding 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. As in the first study, in both male and female rats, a single major metabolite was identified in urine as Bromoxynil phenol. In the 0-8, 8-24 and 24-48-hour 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-hour 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.

Thus, it was concluded that under the test conditions used in this second key study, 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 either 2 or 20 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, liver and kidneys, with no radioactivity being detected in the thyroids. In females treated with 2 mg/kg bw of radiolabelled test material, concentrations in all tissues were significantly higher than in males but similarly distributed. Further smaller amounts however were also detected, e.g.., in thyroid and ovaries. At the higher dose of 20 mg/kg bw of radiolabelled test material the mean concentration of radioactivity in plasma of the females was found to be several fold higher than that found in any other tissues; this was followed by the liver, the thyroids and the kidneys (3.872 μg/g). Whole-body autoradiography of the females 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 phenol 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.

 

The dermal absorption of the test substance was investigated in the rat in a GLP compliant study for which no information on guideline was given in the report (M-283357-01-1, 1985). However, the testconduct is in principle compliant to the OECD test guideline 427 as adopted 2004.On the day before the administration, the male rats were surgically implanted with an in-dwelling cannula in the right external jugular vein under anaesthesia and the hair on their backs was clipped so that a skin area of 25 cm² was exposed. A neoprene rubber template was glued around the treatment area. The rats received single dermal applications of the radiolabelled test material at doses of 1037μg/25 cm2 of skin (acetone used as vehicle), 137μg/25 cm2 of skin (water as vehicle) and 67μg/25 cm2 of skin (water as vehicle) for 48 h. These doses corresponded to 46.9, 6.2 and 3μCi, respectively. In fact, the 46.9 µCi group comprised two animals whereas each of the remaining groups consisted of one single animal.The study is considered valid, scientifically acceptable and appropriate for the assessment of dermal absorption in the rat, despite of the low number of animals used. Treatment was followed by a recovery period and the animals were finally sacrificed after 96 h post-treatment.Sampling was performed during the exposure period of 48 h and for 48 h after the skin wash. For skin wash of the application site, a 1:1 mixture of acetone/water was used and the wash was collected and analyzed for radioactivity together with the wrapping. Urine and feces were collected daily during the exposure period and for 48 h thereafter. Blood was withdrawn from the cannulae at 0.5, 1, 2, 4, 8, 12, 24, 32 and 48 h postdose application and at the same periodicity following the skin wash. Expired volatiles were trapped in acetonitrile and collected at 8, 24 and 48 hours post-application and at 8 h post-washing. Expired CO2 was collected in ethanolamine/2-methoxyethanol traps (3:7 v/v) at 8, 24 and 48 hours post-dose application and following the same schedule following the skin wash. 48 hours after removal of the wrapping, the animals were sacrificed under Metofane anesthesia. Quantification of radioactivity was performed by liquid scintillation counting either by direct addition of the sample to a suitable scintillation fluid or after pre-treatment. Pre-treatment consisted of solubilisation using sodium hydroxide. The total recovery of radioactivity relative to the administered dose was 88.5 and 91.2% for the acetone vehicle and 95.7 and 98.4% for the water vehicle. Comparable to the basic toxicokinetics studies, the predominant route of excretion was the urine. Most of the remaining radioactivity in the animal was found in the carcass and the washed skin. From the analysis of the blood plasma over the course of the study, the blood plasma radioactivity peaked in the 12 – 32 h period for animals that received the test substances in acetone and in water. In one animal receiving with the test substances in the water vehicle, a plateau in blood plasma radioactivity was observed during the 12 to 24 hour and 24 to 32 hour period. Radioactivity in the plasma after washing the dosing solution from the skin decreased slowly and similarly for all animals dosed with the radiolabelled test material in both vehicles. Expired air accounted for less than 0.2% of the recovered radioactivity.  From the animal tissue and excreta, 40 – 46% of the radioactivity was recovered, while radioactivity recovered from the application materials and in the fluid used to wash the skin was 42.6 and 51.2% of the administered dose for the acetone vehicle and 50.7 and 56.8% for the water vehicle. Absorption, excretion and pharmacokinetic during the exposure and after administration were similar using either acetone or water as vehicle.

Two further studies are available investigating the toxicokinetic characteristics of the test compound and related substances (M-283324-01-1, 1982 and M-262286-01-1, 1984). For both, no information on guideline is available from the study reports, and no robust study summaries were included in the IUCLID data set. These studies however, are included here for purpose of data completeness.

In the first of these two studies, (M-283324-01-1, 1982) which was GLP compliant, three groups of four male rats each received a single oral dose of 10 mg/kg bw of either Bromoxynil phenol or its esters (CAS 1689-99-2 and CAS 3861-41-4). All 3 substances were 14C-radiolabelled. An additional male animal received the vehicle only (corn oil). Urine, feces, volatile gases and 14CO2 were sampled 3, 6, 12, 24, 36, 48, 72 and 96 hours post-dose. Two animals from each group were sacrificed at 24 and 96 h post-dose. Organs and tissues collected at these time points included: blood, liver, kidneys, brain, muscle, fat, gastrointestinal tract, heart, lungs and carcass. All substances were well absorbed (> 80% of initial applied dose) and then excreted via the urine, which was the major route of elimination (> 27 - 50% dose by 24 hours post administration). After 96 hours, > 90% of the dose was eliminated for all three substances. For all substances, <0.1% of the administered dose was found in expired air. Absorption and tissue distribution were observed for all substances with liver, blood, kidney, lung and heart being the most affected. However, the test compounds showe no specific affinities for any organ or tissue in the male rat. The 24-hour tissue concentrations showed no large variations from tissue to tissue suggesting that relatively equal tissue distribution of each of the test compounds occurs in the male rat 24 hours following a single oral dose. The most well absorbed substance was the Bromoxynil phenol, followed by the esters (CAS 1689 -99-2 and CAS 3861-41-4), as seen by tissue distribution of radioactivity as well as the elimination via feces.

In the second study (M-262286-01-1, 1984) which was not GLP compliant, twelve female rats received the test substance orally at a dose level of 20 mg/kg bw. Six animals each were sacrificed 3 or 7 days after dosing. The liver, kidneys, thyroid, muscle, skin and fur, and blood were sampled. The study focused on the tissue distribution of the radioactivity and the identity of the metabolites in the tissues. The plasma protein binding experiment indicated that 99.2% and 99.6% of the radioactivity was bound to plasma proteins in the 3- and 7- day plasma samples, respectively. These results may explain the relatively long half-life of plasma radioactivity in both female and male rats. The component that was detected predominantly in plasma, liver, kidney, skin and fur and muscle was identified as Bromoxynil phenol (87-95%) both sampling time points. A minor component, which was probably a conjugate of the main metabolite, was only detected in the liver (1.9 % radioactivity) and kidney (0.3 % radioactivity) at 3 days. In the 7-days samples, Bromoxynil phenol accounted for 56.4% of the radioactivity in extracts of the thyroids, while it accounted for ca. 95% of the radioactivity found in the thyroids after 3 days. However, only low radioactivity was detected in the thyroid sample after 7 days, hence an underestimation of the amount Bromoxynil phenol could be possible. The more polar minor component accounted for 4.0 %, 0.8 % and 14.1 % of the radioactivity in liver, kidneys and thyroids, respectively (after 7 days) and was not detected in other tissues. Taken together, the results regarding absorption, excretion and tissue distribution are in line with the findings of the other studies investigating the test substance. Absorption was almost complete and Bromoxynil phenol was identified as major metabolite and mainly recovered in the urine.

 

Conclusion and assessment of the toxicokinetic behavior of the test substance

In accordance with Regulation (EC) 1907/2006, Annex VIII, Column 1, Item 8.8 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), assessment of the toxicokinetic behavior of the test substance was conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physicochemical properties. The test substance is a white to slightly yellow coarse powder with a water solubility of 0.03 mg/L at 20°C and pH 7, a molecular weight of 403.11 g/mol and a vapor pressure < 1.0 E-07 Pa. The octanol/water partition coefficient (log Pow) was determined around 5.9 at 25 °C. The water solubility and log Pow are not pH dependent.  

  

Absorption

In general, absorption of a substance depends on the potential to cross biological membranes, which is determined by the molecular weight, the log Pow and water solubility. Mostly, substances cross the membranes by passive diffusion, which requires sufficient solubility in water and lipids, a capability, which is described by the log Pow. In general, log Pow values between -1 and 4 are favorable for absorption whereas ionic substances are thought not to readily diffuse across biological membranes. Chemicals that do not offer these properties may be absorbed via active processes including facilitated diffusion, active transport or pinocytosis (ECHA, 2017).

 

Oral:

In general, molecular weights below 500 and log Pow values between -1 and 4 are favorable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (>1 mg/L). However, lipophilic compounds (log Pow > 4) with low water solubility (< 1 mg/L) as the test substance may also be taken up by micellular solubilisation. Moreover, the substance is rapidly hydrolyzed into Bromoxynil phenol. This substance has a log Pow of 0.38 at pH 7, (1.85 at pH 5, -1.57 at pH 9, all at 23 °C), a molecular weight of 276.9 g/mol and a water solubility of 38 g/L at pH 7 and 20 °C (0.073 g/L at pH 4 and 33 g/L at pH 9). Therefore it is assumed, that the parent compound will be absorbed from the gastrointestinal tract after having been hydrolysed into Bromoxynil phenol. This assumption is further supported by the systemic toxicity, observed after oral administration of the test substance. Furthermore, the mortalities noticed in rats after single oral administration of the test substance, also provide evidence that absorption has occurred. In addition, toxicokinetic studies are available, which showed that the test compound is well and rapidly absorbed from the intestinal lumen in rats, with Bromoxynil phenol being identified as major metabolite.

 

Dermal:

To enable dermal absorption, the substance first has to penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network (ECHA, 2017). The stratum corneum provides the first barrier against hydrophilic compounds and dermal uptake of substances with poor lipophilicity (log Pow <0) will be impeded. With a log Pow of > 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high (ECHA, 2017). The log Pow of the test substance is 5.9 – 6.7, therefore it is probably taken up well by the stratum corneum.

The dermal absorption study (M-283357-01-1) suggests a dermal absorption of 40 – 46% of the administered dose, which is lower than gastrointestinal uptake. Data on acute dermal toxicity (M-185070-01-2) of the test substance showed no acute dermal toxicity to rats, which supports the conclusion that dermal absorption of the test compound is low as compared to the oral route, especially since single oral application of the test substance led to systemic effects and mortalities in rodents. An in vivo skin irritation study on rabbits (M-185075-01-2) revealed that the test compound is not irritating to the skin while a sensitization study (M-185084-01-2) found the test substance to be a skin sensitizer in guinea pigs. Developmental toxicity studies are available, that found an effect of the test substance on rat and rabbit development after dermal application during gestation (M-227387-01-1 and M-227408-01-1). The effects occurred at higher dose levels than in similar studies that used oral administration. Thus, these data provide evidence for a dermal absorption of the test material, however to a lesser extent as compared to the oral way of absorption.

 

Inhalation:

Substances including gases, vapors, liquid aerosols (both liquid substances and solid substances in solution) and finely divided powders/dusts may be absorbed directly from the respiratory tract or, through the action of clearance mechanisms, may be transported out of the respiratory tract and subsequently be swallowed which might lead to absorption in the gastrointestinal tract (ECHA, 2017). In general, substances with a low vapor pressure of < 500 Pa are not favorable for respiratory absorption as those substances are not available for inhalation as vapor (ECHA, 2017). The test substance has a low vapor pressure of 2.4 x 10-5 Pa at 25 °C and thus being of low volatility. However, it has a high log Pow values (> 5) and a low water solubility (< 0.1 mg/L) and may be taken up by micellular solubilization and may penetrate the respiratory tract. In addition, inhalation of aerosols is likely since adverse effects and mortality were observed when the test substance was tested in an acute inhalation study in rats (M-226980-01-1). Therefore, the test substance is considered to be absorbed along the respiratory tract.

 

Distribution

In male and female rats, the test substance was widely distributed over various organs and was especially found in blood (plasma Tmax 7 h), liver, kidney and – for females – in the thyroid. Levels in all tissues and in blood decreased over the observation period. In females, a higher proportion of the test substance (or its metabolites) was detected 168 hours post dosing compared to males (up to circa 8% vs below 2% in males).

 

Excretion

Most of the excretion happens via urine (in rats, within 72 hours or 96 hours in males and females, respectively). Thus, urine was identified as the major route of excretion, accounting for >= 80% of elimination of the initially uptake dose. Repeated oral dosing did not lead to different excretion profiles between the sexes. As compared to urine, excretion via the feces is minor.

 

Metabolism

The metabolization rate of the test substance in the rat is high, as the parent compound was only identified in feces, in small amounts (< 10%). The main metabolic pathway was identified as hydrolyzation of the test material to Bromoxynil phenol. Thus, Bromoxynil phenol is the major metabolite found in urine and feces (ca. 85%). Further identified metabolite accounted for ca. 12.5% and were identified as sulphate and glucuronide conjugates which all yield Bromoxynil phenol after rapid enzymatic hydrolysis.