mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2000 to 26 January 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Objective of study:

distribution

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 860.1300

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

EC Number:

240-539-0

EC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Cas Number:

16484-77-8

Molecular formula:

C10H11ClO3

IUPAC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Test material form:

solid

Remarks:

Cream solid

Radiolabelling:

yes

Remarks:

Goat

Test animals

Species:

other: Goat

Strain:

other: British Saanen

Details on species / strain selection:

The test material is an established broad leaf herbicide. During and following its use residues may result in, or on, plant material destined to be fed to food producing animals. It is necessary, therefore, as part of the safety evaluation, to investigate the extent to which these residues may be transferred to animal tissues and milk, which may be destined for human consumption and also to establish the nature of any transferred residues. This study was designed to investigate the disposition and metabolic fate of [14C]-test material in the lactating goat following multiple oral administration.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 78.8 kg and 82.2 kg on arrival.
- Housing: On the day prior to the start of the study, the animals were transferred to metabolism crates suitable for the separate collection of urine and faeces.
- Diet: The animals were offered hay ad libitum and the amount consumed each day measured. In addition, the animals were fed protein concentrate rations at an approximate rate of 1 kg per day (500 g at each time of milking).
- Water: Ad libitum mains tap water.
- Acclimation period: The goats were acclimatised to the experimental unit for a period of 7 days prior to dosing. The animals were milked twice daily at ca. 0830 h and ca. 1630 h. The animals were weighed during the acclimatisation period, the last occasion being 24 h prior to the first dose administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 34 - 78 %
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Low Dose Formulation (5 ppm):
The dose solution was prepared by weighing 6.90 mg of [14C]-test material and 89.32 mg of non-radiolabelled test material into a 5 mL capacity volumetric flask which was made up to volume with acetone. Sub-samples (3 x 10 μL) of this dose solution were transferred to 10 mL volumetric flasks and made up to volume with acetone. Duplicate aliquots (100 μL) were taken from each flask for LSC analysis to determine the concentration of the test material in the dose solution. The specific activity of the [14C]-test material in the dose solution was determined as 9.751 μC.mg^-1. Aliquots of the dose solution (volume calculated from concentration of dose solution) were then dispensed into 14 capsules (272 μL per capsule) and the solvent allowed to evaporate in a flow of air. A further 2 aliquots were taken from the dose solution (one at the beginning of capsule dispensing and one at the end), and made up to 20 mL with acetone. Duplicate samples from these aliquots (25 μL) were subsequently analysed by LSC to determine the amount of radioactivity applied to each capsule.
- High Dose Formulation (50 ppm):
The dose solution was prepared by weighing 73.14 mg of [14C]-test material and 953 mg of non-radiolabelled test material into a 5 mL capacity volumetric flask, which was made up to volume with acetone. Sub-samples (3 x 10 μL) of this dose solution were transferred to 10 mL volumetric flasks and made up to volume with acetone. Duplicate aliquots (25 μL) were taken from each flask for LSC analysis to determine the concentration of the test material in the dose solution. The specific activity of the [14C]-test material in the dose solution (volume calculated from concentration of dose solution) was determined as 9.357 μC.mg^-1. Aliquots of the dose solution were then dispensed into 15 capsules (255 μL per capsule) and the solvent allowed to evaporate in a flow of air. A further 2 aliquots were taken from the dose solution (one at the beginning of capsule dispensing and one at the end) and made up to 100 mL with acetone. Duplicate samples from these aliquots (25 μL) were subsequently analysed by LSC to determine the amount of radioactivity applied to each capsule.

All the capsules were capped and stored at ca +4 °C until dosing.
A [14C]-test material radiochemical purity check was also carried out on the pre-dose formulation for confirmation prior to dosing. A radiochemical purity value of 97.0 % was determined by TLC analysis using the same method that was used for the dose stability checks.

Duration and frequency of treatment / exposure:

The daily dose for each goat was administered over 7 consecutive days in 2 equal portions, one after morning milking (ca. 0830 h) and one following afternoon milking (ca. 1630 h). Goat 1 was dosed at the low dose level and Goat 2 at the high dose level.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 ppm

Remarks:

Low dose level - 100 µCi per day

Doses / concentrations 2

Dose / conc.:

50 ppm

Remarks:

High dose level - 1000 µCi per day

No. of animals per sex per dose / concentration:

Two females

Control animals:

no

Details on dosing and sampling:

The appearance and behaviour of the animals were monitored daily.

Urine and faeces were collected during the day prior to the first dose administration and at intervals of 24 h following administration of the first daily dose until sacrifice. The cages were rinsed with water at each collection time and the rinses retained.
Milk samples were collected from each animal in the morning prior to administration of the first dose and then twice daily throughout the study period, immediately prior to the morning (ca. 0830 h) and afternoon (ca. 1630 h) doses. The final milk collection was made immediately prior to sacrifice.
The weights of urine, faeces, cage wash and milk samples were recorded and total radioactivity measured.
Approximately 23 h after administration of the final dose, the goats were stunned using a captive bolt, pithed and exsanguinated by severance of the major neck vessels. The following biological fluids and tissues were removed and assayed for total radioactivity:
Whole Blood, Omental fat, Plasma, Renal fat, Kidneys, Skeletal muscle (maximum amounts of hind and fore quarter), Liver, Gastrointestinal Tract, Gastrointestinal Tract Contents.

Total radioactivity was measured in all samples collected except for the gastrointestinal tract and contents, which were collected as a precautionary measure.
Haematology and clinical chemistry parameters were measured immediately prior to sacrifice to back up daily animal observations. Although there was some degree of variation between the goats, the haematology and clinical chemistry parameters were within the normal range of values expected for goats.

Storage of Biological Samples
Biological samples were analysed on the day of collection wherever possible, prior to storage at ca. -20 °C. Where immediate analysis was not possible, biological samples were stored at ca. -20 °C prior to and following analysis. The longest frozen storage interval, of 2 - 3 months, was for the tissue samples (from initial extraction to HPLC analysis). Since metabolite profiles for the kidney methanol/water and enzyme hydrolysis extracts were similar, this indicates that the [14C]-residues in the tissues are stable over the longest storage interval.

DETERMINATION OF RADIOACTIVITY
Liquid Scintillation Counting (LSC)
All samples prepared in scintillant were counted for 5 min using a Packard 1600 TR Liquid Scintillation Analyser (Canberra Packard Limited) with automatic quench correction by external standard method. All biological samples were processed in duplicate. Scintillation vials were allowed to heat and light stabilise prior to analysis. Representative blank sample values were subtracted from sample count rates to give net d.p.m. per sample. A limit of quantification of 30 d.p.m. above background has been instituted in these laboratories. If results arose from data less than 30 d.p.m. above background, the fact is so noted in the results section of the report. 30 d.p.m. is typical of the background count obtained throughout the study.

Combustion Analysis
Samples for combustion were weighed into Combustocones® (Packard Instruments Company Limited) and combusted using a Model 306 or 307 Tri-Carb Automatic Sample Oxidiser (Canberra Packard Limited). The resultant 14CO2 was absorbed in Carbo-Sorb® and mixed automatically with Permafluor®E+ scintillation fluid. The efficiency of the combustion process was routinely checked several times throughout each production run by combusting quality control standards (Spec-ChecTM-14C). Combustion efficiency was shown to be greater than 97 % throughout the experimental period.
Preparation of Samples for LSC
Urine and cage wash
Duplicate samples of urine (ca. 0.9 g) and cage wash (ca. 0.9 g) were mixed with 10 mL Quickszint 1® (Zinsser) scintillation fluid prior to scintillation counting.

Plasma
Whole blood was centrifuged at ca. 3 500 rpm for 10 min and plasma and blood cells separated. Duplicate aliquots of plasma (ca. 0.5 g) were mixed with 10 mL Quickszint 1® (Zinsser) scintillation fluid prior to LSC.

Whole Blood
Duplicate samples of whole blood (ca. 0.3 g) were combusted as described previously.

Milk
Duplicate ca. 4 g aliquots were mixed with 10 mL Quickszint scintillation fluid to form a gel, then analysed by LSC.

Faeces
Faeces samples were homogenised in 3 mL/g of acetronitrile:water (50:50, v/v) using a Silverson overhead homogeniser. Samples of each homogenate (2 x ca. 0.3 g) were taken for combustion as described previously.

Liver and Kidney
Liver and kidney samples were allowed to thaw prior to homogenising in a Waring Blender. Duplicate homogenate samples (ca. 0.3 g) were taken for combustion as described previously.

Muscle
Muscle samples were initially processed frozen using a Hobart mincer then homogenised with dry ice using a Hobart blender to produce a fine powder. Samples of each homogenate (2 x ca. 0.3 g) were taken for combustion as described previously.

Omental and Renal Fat
Omental and renal fat samples were chopped into small pieces while partially frozen and then homogenised with dry ice using a Hobart blender to produce a fine powder. Duplicate homogenate samples (ca. 0.5 g) were taken for combustion as described previously.

EXTRACTION OF TISSUES, MILK, FAECES, AND URINE
Based on the residue levels, samples from Goat 2 were selected for extraction and chromatographic analysis. General procedures for the extraction and processing of samples prior to chromatographic analysis were as follows.
Each sample (except urine and milk) was extracted with solvent (3 mL/g) by maceration using an overhead Silverson macerator. Following each extraction, the solvent extract was separated from the post-extracted solid (PES) by centrifugation (3 000 rpm; 10 min) and decanting. Sub-samples of the PES were submitted for combustion analysis. The volume of individual and/or combined extracts was measured and aliquots analysed by liquid scintillation counting (LSC). Total combined extracts were concentrated under nitrogen. The final volume of concentrated extracts was measured and aliquots analysed by LSC. Details of additional procedures for different sample types are as follows:

Liver and kidney
Liver and kidney were processed using the same methods, except that the kidney enzyme aqueous extract was further processed for HPLC analysis following methanol precipitation, concentration and centrifugation. A number of stages were involved in the extraction process:
Approximately 30 g of liver and kidney were extracted by maceration on 2 occasions with methanol (3 mL/g) followed by one occasion with 3 mL/g of methanol:water (9:1, v/v). Between extractions, each sample was centrifuged (3 000 rpm; 10 min) and the supernatant removed by decanting. The supernatants were combined, the total volume was measured and aliquots removed for LSC. Supernatants were concentrated under nitrogen following hexane partitioning (3 x supernatant volume) to remove fat. Total volumes were measured and aliquots removed for LSC. The hexane extracts were not processed further, since all the radioactivity was quantitatively accounted for in each methanol layer following partitioning.
The PES from each liver and kidney sample was further extracted by mixing with approximately 2 g protease enzyme (Type 1: Crude, from bovine pancreas) and ca. 25 mL of 0.1 M potassium phosphate buffer (pH 7.4) and then homogenised using a Silverson overhead homogeniser. The sample was agitated at 36 °C for ca. 48 h in a water bath then centrifuged (10 000 r.p.m., 30 min). Duplicate aliquots of the supernatant were taken for LSC.
The PES remaining after protease extraction was then further extracted by mixing with approximately 2 to 3 g of pepsin (from porcine stomach mucosa) and then homogenising with 0.1 M hydrochloric acid (50 or 75 mL) using a Silverson overhead homogeniser. The sample was agitated at 37 °C for ca. 48 h in a water bath then centrifuged (4 000 rpm, 30 min). Duplicate aliquots of the supernatant were taken for LSC.
The activity of the pepsin enzyme was confirmed by hydrolysis of bovine haemoglobin to trichloroacetic acid soluble peptides (37 °C, pH 2) using u.v. spectrophotometry at 280 nm. The activity of the protease enzyme was confirmed by hydrolysis of N-t-BOC-L-Glutamic acid Phenyl Ester Substrate Solution (prepared by dissolving N-t-BOC-L-Glutamic acid Phenyl Ester in 1,4- dioxane) to N-t-BOC-L-Glutamic acid and phenol (37 °C, pH 7.8) using u.v. spectrophotometry at 270 nm.
The PES remaining after pepsin extraction was subjected to base hydrolysis in 3N sodium hydroxide and methanol (1:4:1, weight PES: ml base: mL methanol) at approximately 58 °C overnight in a shaking water bath. Following hydrolysis, the sample was adjusted to pH 2 using HCl and then filtered. The volume of filtrate was measured and aliquots removed for LSC. The remaining PES was further hydrolysed as above except that the 3N sodium hydroxide: methanol ratio was 1:1, the temperature of hydrolysis was increased to 70 °C and the time extended to 2 days. The sample was centrifuged (3 000 rpm, 15 min), the volume of supernatant measured and aliquots removed for LSC.

Milk
The 56 h and 152 h milk samples were extracted using the same methods. An approximately equal volume of acetonitrile was added to 50 mL of milk and the sample mixed by shaking and vortexing. The sample was centrifuged (4 000 rpm, 10 min) and aliquots of the supernatant were removed for liquid scintillation counting (LSC). Following removal of the acetonitrile by rotary evaporation at 30 °C, the aqueous phase was partitioned against diethylether (1 x 150 mL) followed by hexane (2 x 150 mL) to remove fatty material. The organic phases were not processed further, since all the radioactivity was quantitatively accounted for in the aqueous layer following partitioning. Aliquots of the aqueous phase were removed for LSC. The aqueous sample was concentrated under nitrogen and aliquots of the concentrated sample removed for LSC. The concentrated aqueous extract was centrifuged at 4 000 rpm for 10 min and the pellet back washed with acetonitrile (2 x 2 mL) by vortex mixing followed by centrifugation (4 000 rpm, 10 min) to recover radioactivity losses.
The supernatants from the back washes were combined with the supernatants of the concentrated aqueous extract and subsequently concentrated under nitrogen and centrifuged (4 000 rpm, 10 min). Duplicate aliquots were analysed by LSC.
Extraction efficiencies were calculated by comparing the levels of radioactivity remaining at key stages of processing with those determined following direct LSC of the milk samples prior to processing.

Faeces
For Goats 1 and 2 (48 h and 168 h) faeces samples, approximately 20 g of sample was extracted on 3 occasions with methanol (3 mL/g) by maceration as detailed previously. The extracts were combined and aliquots removed for LSC.
Goat 1 faeces (48 h and 168 h) combined methanol extracts were partitioned with hexane (3 x equal volume) to remove fatty material prior to concentration of the extracts. The Goat 1 (hexane partitioned methanol extracts) and Goat 2 (methanol extracts) were evaporated under nitrogen to a smaller volume and a sub-sample (1 mL) was centrifuged at 10 000 rpm for 5 min. Duplicate aliquots of the sub-sample supernatant were taken for LSC prior to HPLC analysis.

Urine
Sub-samples (1 mL) of 48 h and 168 h urine from Goat 2 were centrifuged at 10 000 rpm for 5 min (Jouan A14 centrifuge) to remove particulate material prior to chromatography. Urine samples (40 mL) from Goat 1 (48 h and 168 h) were concentrated under nitrogen to a lower volume (17 mL). In each case, duplicate aliquots of the supernatant were taken for LSC prior to HPLC analysis.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HPLC was performed using the following equipment and conditions:

HPLC Model:
HP 1100 Series Modules Liquid Chromatograph
HP 1100 Series Variable Wavelength Detector
HP 1100 Series Vacuum Degasser
HP 1100 Series Quaternary Pump
HP 1100 Series Thermostatted Autosampler
HP 1100 Series Thermostatted Column Compartment
Radiodetector Model: Radiomatic™ Flo-one®\Beta, Flow Scintillation Analyser (Model 150TR)
Fraction Collector: Gilson Model 202 Fraction Collector and fractions collected every minute
Conditions:
Column: YMC ODS-AQ (250 x 4.6 mm, 5 μm)
Guard Column: Phenomenex Security Guard (10 x 4.6 mm, 5 μm)
Mobile Phase A: 1 % Ammonium acetate in water
Mobile Phase B: 1 % Ammonium acetate in methanol

Gradient:
0 min: 90 % A, 10 % B
40 min: 10 % A, 90 % B
50 min: 10 % A, 90 % B
55 min: 90 % A, 10 % B
Flow Rate: 1.0 mL/min
Column Temperature: Ambient
u.v. Detector Wavelength: 254 nm
Scintillant: Ultima-Flo™M

Sample Analysis
Non-radiolabelled test material analytical reference standard was prepared in methanol:water (1:1, v/v) at a concentration of ca. 1 mg/mL. Sub-samples of sample extracts and urine were mixed with non-radiolabelled test material for accurate comparison of retention times.
For the urine samples, analysed with on line radiodetection, data were captured using the Multichrom system and quantification carried out by peak integration. The sample extracts were analysed using fraction collection/LSC as a means of radiodetection.
Fractions containing less than background levels + 30 d.p.m. of radioactivity were not included in further calculations. Background levels of radioactivity were calculated as the mean of the values for the first two fractions in each analytical run (i.e. void volume of the HPLC column).
During HPLC analysis, the recovery of radioactivity from the HPLC column was checked by quantifying the radioactivity eluted from the system and comparing this with the level injected. Column recoveries were determined for one of each sample type.

HPLC column recoveries for Goat 2 liver, kidney enzyme hydrolysis extract, 152 h milk, 48 h faeces, 168 h faeces, 48 h urine and 168 h urine were 110.0 %, 112.6 %, 155.7 %, 96.5 %, 97.5 %, 101.6 % and 96.8 %, respectively, demonstrating acceptable recovery of injected radioactivity. In some samples the column recoveries are high due to the low levels of radioactivity injected.

LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY (LC-MS)
HPLC Model: HP 1090 Series Liquid Chromatograph
Mass Spectrometer Model: Finnigan MAT TSQ 7000 Mass Spectrometer
Radiodetector Model: Packard 150TR Radiochemical Detector
Data Handling: Xcalibur Software Version 1.1

HPLC Spectrometer Conditions
Guard Column: Phenomenex Security Guard (10 mm x 4.6 mm, 5 μm)
Column: YMC ODS-AQ (250 mm x 4.6 mm, 5 μm)
Mobile Phase A: 0.5 % Ammonium acetate solution
Mobile Phase B: 0.5% Ammonium acetate in acetonitrile
Gradient:
0 min: 90 % A, 10 % B
40 min: 10 % A, 90 % B
50 min: 10 % A, 90 % B
55 min: 90 % A, 10 % B
Flow rate: 1 mL/min
Split: ca. 100 - 200 μL/min to mass spectrometer
Injection Volume: 5 - 100 μL

Mass Spectrometer Conditions
The following types of mass spectrometer techniques were employed for the analysis of samples:

A) Full Scan Analysis
Ionisation Mode: Negative ion electrospray ionisation
Spray Voltage: 4.5 kV
Capillary Temperature: 210 °C
Sheath Gas: 70 psi
Auxiliary Gas: 5 units
APICID Offset: 10 V
Scanning Mode: 50 – 500, scan rate 1 sec

B) Product Ion Scan Analysis
Collision Gas: Argon
Collision Gas Pressure: ca. 2 mTorr
Collision Offset: 15 V
Parent Ion: m/z 213
Product Ion Range: m/z 50 - 220
Scan Time: 1 sec

C) Multiple Reaction Monitoring
Collision Gas: Argon
Collision Gas Pressure: ca. 2 mTorr
Collision Offset: 15 V
Transition 1
Parent Ion: m/z 213
Product Ion: m/z 141
Scan Time: 0.5 sec
Transition 2
Parent Ion: m/z 215
Product Ion: m/z 143
Scan Time: 0.5 sec

CALCULATIONS
Total radioactivity data were collected and calculated using the validated Debra 5.2a computerised data acquisition system.
The following principles were used in the calculation of data within this report:

Total radioactivity data
Specific activity of [14C]-test material in the dose solutions:
Low Dose Level = 9.751 μCi/mg
High Dose Level = 9.357 μCi/mg

Calculated daily dose (mg) = ((mean total d.p.m. in a dose capsule / 2.22x 10^6 (d.p.m./µCi)) / Specific activity) x 2

Total dose administered (d.p.m.) = Mean total d.p.m. in a dose capsule x 14 doses

Total dose administered (mg) = (Total d.p.m. administered / 2.22 x 10^6 (d.p.m./µCi)) / Specific activity (µCi/mg)

Calculated dose level (p.p.m.) = Calculated daily dose (mg/day) / Mean food consumption on study (kg/day)

Total d.p.m. in samples = Mean d.p.m/g x Total Sample Weight (g)

μg equiv/g = Mean d.p.m.6^-1 in sample / specific activity (d.p.m/µg)

% Administered dose in samplle = (Total d.p.m. in sample / Total d.p.m. in administered dose) x 100

Chromatography data
Total radioactive residue (TRR) = Total d.p.m. in extract + P.E.S.

%Initial extraction efficiency (%TRR) = (Total d.p.m in extract / Total d.p.m. in extract + P.E.S) x 100

% Final extraction efficiency (% TRR) = % initial extraction efficiency (% TPR) x (Overall procedural recovery / 100)

Extraction efficiency (ppm) = (Total d.p.m. in extract / Total d.p.m. in extract + P.E.S) x ppm in sample.

% Final extraction efficiency (ppm) = %Extraction efficiency (ppm) x (Overall procedural recovery / 100)

Metabolite (%TPR) = % total peaks / 100) x % final extraction efficiency (TPR).

Metabolite (ppm) = (% total peaks / 100) x % final extraction efficiency (ppm)
Limit of detection (LOD) = Background (d.p.m.)
Limit of quantification (LOQ) = Background (d.p.m.) + 30 d.p.m.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:

Nominal 5 ppm Dose Level, Goat 1
The highest residue levels in tissues at the time of killing (23 h post last dose) were recorded in the kidney, which accounted for only 0.007 μg equiv/g (0.002 % dose).
Levels in the liver, renal fat and omental fat did not exceed 0.001 μg equiv/g and concentrations in hind and fore muscle were below the limit of reliable determination.
Concentrations of total radioactivity in the whole blood and plasma were also low at 0.005 and 0.004 μg equiv/g, respectively. The amount of total radioactivity recorded in tissues represented <0.01 % of the administered dose. By the end of the study period the total recovery of radioactivity in all samples accounted for 97.3 % of the administered dose.

Nominal 50 ppm Dose Level, Goat 2
The highest concentration of total radioactivity found in tissues was in kidney at 0.097 μg equiv/g (0.002 % of the administered dose). The concentration of total radioactivity in liver was 0.031 μg equiv/g and this accounted for 0.005 % of the dose.
Levels of radioactivity in the omental and renal fat were 0.003 μg equiv/g and concentrations in the hind muscle and fore muscle were very low at 0.001 μg equiv/g.
Concentrations of total radioactivity in the whole blood and plasma were 0.029 and 0.035 μg equiv/g, respectively.
The amount of total radioactivity recorded in tissues represented < 0.01 % of the administered dose. The total recovery of radioactivity in all samples at the end of the study period accounted for 95.9 % of the administered dose.

Goat 2 (High Dose) Liver and Kidneys
Liver and kidney samples from Goat 2 were processed using a sequential extraction approach. Each sample was initially extracted with methanol, followed by methanol/water (9:1, v/v). Aqueous methanol extraction was followed by protease enzyme extraction and then by pepsin enzyme extraction of the post-extracted solid (PES). Each remaining PES was then base hydrolysed with 3M NaOH in methanol, initially at 58 °C for 20 h and then at 70 °C for 48 h in a shaking water bath. The combined methanol/water extracts were partitioned with hexane and then concentrated and centrifuged prior to HPLC analysis. Goat 2 kidney pepsin enzyme aqueous extracts were further processed for HPLC analysis by methanol precipitation followed by concentration and centrifugation of the supernatant.
Initial extraction efficiencies with methanol followed by methanol/water (9:1, v/v) were highest in the liver, with the combined initial extracts representing 58.0 % TRR (0.018 ppm). Kidney combined initial extracts represented 47.7 % TRR (0.046 ppm). Following hexane partitioning to remove co-extracted fat and subsequent concentration and centrifugation of the methanol/water extracts, minor losses of radioactivity were observed such that the final liver and kidney concentrated aqueous/methanol extracts were 54.7 % TRR (0.017 ppm) and 44.3 % TRR (0.043 ppm), respectively.
Protease enzyme hydrolysis recovered a further 5.7 % TRR (0.002 ppm) and 3.5 % TRR (0.003 ppm) from the liver and kidney PES samples, respectively. Following pepsin enzyme hydrolysis a further 20.1 % TRR (0.006 ppm) and 20.2 % TRR (0.020 ppm) was recovered from the liver and kidney PES samples, respectively. Losses of radioactivity from Goat 2 kidney pepsin enzyme processed aqueous extracts were low, with 16.6 % TRR (0.016 ppm) recovered. Both the liver and kidney PES remaining following the enzyme hydrolysis experiments were further hydrolysed with 3M NaOH in methanol as described previously. Levels of 2.7 % (0.001 ppm) and 13.5 % TRR (0.004 ppm) were released following incubation of the liver PES at 58 °C for 20 h and 70 °C for 48 h, respectively. Corresponding levels for the kidney PES were 2.4 % (0.002 ppm) and 13.0 % TRR (0.013 ppm).

Goat 2 (High Dose) Liver
HPLC analysis of the liver aqueous/methanol extracts, containing 0.017 ppm (54.7 % TRR), showed the presence of 1 polar unknown component at a retention time of 5 min.

Goat 2 (High Dose) Kidney
HPLC analysis of the kidney aqueous/methanol extracts, containing 0.043 ppm 44.3 % TRR), showed the presence of 3 components. The most abundant of these represented 0.030 ppm (31.1 % TRR) and was identified as test material by cochromatography. The remaining 2 components were unidentified and represented 0.003 ppm and 0.010 ppm (2.7 % TRR and 10.5 % TRR). The kidney pepsin enzyme hydrolysis extract, containing 0.016 ppm (16.6 % TRR), showed the presence of 1 component corresponding to test material by co-chromatography.

Goat 2 (High Dose) Kidney
The reconstructed ion chromatogram of the ion at m/z 213 contained a peak which corresponded to the small peak in the radiochromatogram with a retention time of 25.21 min. The low intensity of the signal resulted in a spectrum containing numerous background ions. However, the spectrum contained the ion at m/z 213, its chlorine atom associated isotope peak at m/z 215 and the fragment ion at m/z 141. The low amount of material in this sample prevented the generation of a product ion spectrum. However, the mass spectrometer was programmed to monitor the transition from 213 => 141 and 215 => 143, as these transitions were characteristic of the test material. The retention times, the full scan spectrum and the reconstructed ion chromatograms of the transitions from m/z 13 =>141 and m/z 215 => 143 were consistent with those observed for authentic test material. Therefore, this component was characterised as the test material.

Details on excretion:

Nominal 5 ppm Dose Level, Goat 1
Following 14 twice daily oral administrations of [14C]-test material at a nominal dose level of 5 ppm of daily food consumption per day, the major route of excretion was via urine, with 81.0 % of the administered dose accounted for by the end of the study period (23 h post last dose). Excretion via faeces over the same time period accounted for 11.0 % of the dose.
Radio-HPLC analysis of urine and faeces indicated that the test material accounted for a mean of ca. 96.4 % of the radioactivity present in urine and a mean of ca. 90.3 % of the radioactivity present in faeces. This indicates that at least 88.0 % of an oral dose of [14C]-test material was excreted as parent molecule at the low dose level.
Residues in milk were low at all timepoints, with levels never rising above 0.001 μg equiv/g. Residues in milk in total accounted for 0.019 % of the administered dose.

Nominal 50 ppm Dose Level, Goat 2
Following 14 twice daily oral administrations of [14C]-test material at a nominal dose level of 50 ppm of daily food consumption per day, the main route of excretion was via urine with 64.5 % of the dose being recovered. Excretion via faeces over the same time period accounted for 24.9 % of the dose.
Radio-HPLC analysis of urine and faeces indicated that test material accounted for a mean of ca. 94.1 % of the radioactivity present in urine and a mean of ca. 92.5 % of the radioactivity present in faeces. This indicates that at least 83.7 % of an oral dose of [14C]-Test material was excreted as parent molecule at the high dose level.
Levels of total radioactivity in milk throughout the study period were low, with a maximum concentration of 0.013 μg equiv/g at 128 and 152 h declining to 0.007 μg.equiv/g at 175 h (8 h post last dose). The total recovery of radioactivity in milk at 175 h post dose accounted for only 0.019 % of the administered dose.

Goat 1 (Low Dose) Faeces and Urine
At the 5 ppm dose level (Goat 1), faeces (48 h and 168 h post dose) were extracted with methanol and the concentrated extracts analysed by HPLC following hexane partitioning and concentration to a lower volume and centrifugation. Two urine samples from Goat 1 (48 h and 168 h) were analysed by HPLC following concentration under nitrogen to a lower volume.
Extraction of faeces (48 h and 168 h) with methanol recovered 95.1 % TRR and 94.7 % TRR. Following hexane partitioning, to remove co-extracted fat, and subsequent concentration and centrifugation of the methanol extracts, some losses of radioactivity were observed such that the final faeces concentrated extracts collected were 95.1 % TRR (48 h) and 87.3 % TRR (168 h), respectively.
No procedural losses were incurred following concentration and centrifugation of urine samples (48 h and 168 h) prior to HPLC analysis.

Goat 2 (High Dose) Faeces and Urine
At the 50 ppm dose level (Goat 2), faeces (48 h and 168 h post dose) was extracted with methanol and the concentrated extracts analysed by HPLC, following concentration to a lower volume and centrifugation. Two urine samples from Goat 2 (48 h and 168 h) were analysed by HPLC following centrifugation to remove particulate matter.
Extraction of faeces (48 h and 168 h) with methanol recovered 97.0 % TRR and 96.7 % TRR, respectively. Following concentration and centrifugation of the methanol extracts, minor losses of radioactivity were observed such that the final faeces concentrated extracts collected at 48 h and 168 h were 97.0 % TRR and 94.6 % TRR, respectively.
There were minor procedural losses following centrifugation of urine samples (48 h and 168 h) prior to HPLC analysis, the samples analysed representing 99.8 % TRR (18.22 % dose) and 97.2 % TRR (9.22 % dose), respectively.

Goat 2 (High Dose) Milk
At the 50 ppm dose level (Goat 2), milk (48 - 56 h and 144 - 152 h post dose) was extracted with acetonitrile, partitioned with diethyl ether and hexane, then concentrated under nitrogen prior to analysis by HPLC. Extraction with acetonitrile recovered 89.8 % TRR and 86.6 % TRR from the 56 h and 152 h milk samples, respectively. Following partitioning with hexane and diethyl ether to remove co-extracted fat and subsequent concentration and centrifugation of the extracts, high losses of radioactivity were observed in both extracts such that the final values were 30.8 % TRR and 29.6 % TRR, respectively. The concentrations of radioactivity in the milk samples were low (0.011 μg.equiv/g and 0.013 μg.equiv/g). It is highly likely that this, as well as the low levels of radioactivity in the concentrated initial extracts, accounts for the low procedural recoveries. Since no or minor procedural losses occurred following the hexane partitioning, no fat soluble metabolites would have been present in the hexane extracts.

Goat 1 (Low Dose) Urine (48 h)
HPLC analysis of the 48 h urine sample, which contained 10.66 % of the total administered radioactivity (100 % TRR), showed the presence of 4 components. The most abundant of these represented 10.33 % of the total administered radioactivity (96.9 % TRR) and was identified as test material by co-chromatography. The remaining 3 minor components were unidentified and represented 0.04 % to 0.16 % of the total administered radioactivity (0.3 % to 1.5 % TRR).

Goat 1 (Low Dose) Urine (168 h)
HPLC analysis of the 168 h urine sample, which contained 13.70 % of the total administered radioactivity (100 % TRR), showed the presence of 4 components. The most abundant of these represented 13.14 % of the total administered radioactivity (95.9 % TRR) and was identified as test material by co-chromatography. The remaining 3 minor components were unidentified and represented 0.15 % to 0.21 % of the total administered radioactivity (1.1 % to 1.5 % TRR).

Goat 1 (Low Dose) Faeces (48 h)
HPLC analysis of the 48 h faeces extracts, which contained 2.23 % of the total administered radioactivity (95.1 % TRR), showed the presence of 2 components. The most abundant of these represented 2.19 % of the total administered radioactivity (93.3 % TRR) and was identified as test material by co-chromatography. The remaining minor component was unidentified and represented 0.04 % of the total administered radioactivity (1.8 % TRR).

Goat 1 (Low Dose) Faeces (168 h)
HPLC analysis of the 168 h faeces extracts, which contained 1.14 % of the total administered radioactivity (87.3 % TRR), showed the presence of 1 component which was identified as Test material by co-chromatography.

Goat 2 (High Dose) Urine (48 h)
HPLC analysis of the 48 h urine sample, which contained 18.20 % of the total administered radioactivity (99.8 % TRR), showed the presence of 4 components. The most abundant of these represented 17.42 % of the total administered radioactivity (95.4 % TRR) and was identified as Test material by co-chromatography. The remaining 3 minor components were unidentified and represented 0.24 % to 0.28 % of the total administered radioactivity (1.3 % to 1.6 % TRR).

Goat 2 (High Dose) Urine (168 h)
HPLC analysis of the 48 h urine sample, which contained 9.22 % of the total administered radioactivity (97.2 % TRR), showed the presence of 7 components. The most abundant of these represented 8.79 % of the total administered radioactivity (92.7 % TRR) and was identified as Test material by co-chromatography. The remaining 6 minor components were unidentified and represented 0.02 % to 0.13 % of the total administered radioactivity (0.2 % to 1.3 % TRR).

Goat 2 (High Dose) Faeces (48 h)
HPLC analysis of the 48 h faeces extracts, which contained 2.46 % of the total administered radioactivity (97.0 % TRR), showed the presence of 3 components. The most abundant of these represented 2.38 % of the total administered radioactivity (93.9 % TRR) and was identified as test material by co-chromatography. The remaining 2 minor components were unidentified and represented 0.02 % and 0.06 % of the total administered radioactivity (0.9 % and 2.2 % TRR).

Goat 2 (High Dose) Faeces (168 h)
HPLC analysis of the 168 h faeces extracts, which contained 3.80 % of the total administered radioactivity (94.6 % TRR), showed the presence of 3 components. The most abundant of these represented 3.66 % of the total administered radioactivity (91.1 % TRR) and was identified as test material by co-chromatography. The remaining 2 minor components were unidentified and represented 0.05 % and 0.09 % of the total administered radioactivity (1.3 % and 2.2 % TRR).

Goat 2 (High Dose) Milk (56 h)
Due to the very low residue levels in the 56 h milk final concentrated extract (30.8 % TRR, 0.003 ppm), no radiolabelled residues were observed as the levels of radioactivity were below the limit of quantification.

Goat 2 (High Dose) Milk (152 h)
HPLC analysis of the 152 h milk final concentrated extract, which represented a residue level of 0.004 ppm (29.6 % TRR), showed the presence of a single unidentified component. There was evidence for the presence of test material at a retention time of 29 min, but at levels less than the limit of quantification.

Confirmation of Test material in Urine, Faeces and Kidney by Mass Spectroscopy
The analysis of test material by negative ion electrospray ionisation liquid chromatography-mass spectrometry (ESI-LC-MS) revealed a peak with a retention time of 25.18 min. The spectrum of this peak contained the ion at m/z 213, which corresponded to the deprotonated molecular ion ([M-H]-). The spectrum also contained an ion at m/z 141.
The product ion spectrum of the deprotonated molecular ion ([M-H]-) at m/z 213, fragmented to form the product ion at m/z 141.
This multiple reaction monitoring approach permitted a specific and sensitive method for the characterisation of the test material at low concentrations.

Sample Analysis
Goat 2 (High Dose) Urine (168 h)
The reconstructed ion chromatogram of the ion at m/z 213 contained a peak which corresponded to the peak in the radiochromatogram with a retention time of 24.80 min. The spectrum contained the ion at m/z 213 and its chlorine atom associated isotope peak at m/z 215. The fragment ions at m/z 141 and 143 were also present in the spectrum. The product ion spectrum of the ion at m/z 213, fragmented to form the ion at m/z 141. The retention time of both full scan spectrum and product ion spectrum for this component were consistent with those observed for authentic test material. Therefore, this component was characterised as the test material.

Goat 2 (High Dose) Faeces (168 h)
The reconstructed ion chromatogram of the ion at m/z 213 contained a peak which corresponded to the peak in the radiochromatogram with a retention time of 24.98 min. The spectrum contained the ion at m/z 213 and its chlorine atom associated isotope peak at m/z 215. The fragment ions at m/z 141 and 143 were also present in the spectrum. The product ion spectrum of the ion at m/z 213, fragmented to form the ion at m/z 141. The retention time, full scan spectrum and product ion spectrum of this component were consistent with those observed for authentic test material. Therefore, this component was characterised as the test material.

Metabolite characterisation studies

Metabolites identified:

not measured

Any other information on results incl. tables

Summary of the Distribution, Excretion and Recovery of Administered Radioactivity.

 

Summary of Cumulative† Total Radioactivity Data for Goats Expressed as μg equiv/g and % Total Dose Administered
Sample	5 ppm Goat 1		50 ppm Goat 2
	μg equiv/g	% Dose	μg equiv/g	% Dose
Urine	NA	80.96	NA	64.53
Faeces	NA	10.97	NA	24.86
Cage wash	NA	5.31	NA	6.52
Milk	NA	0.02	NA	0.02
Omental fat	*0.001	NA	*0.003	NA
Renal fat	*0.001	NA	0.003	NA
Kidney	0.007	<0.01	0.097	<0.01
Liver	*0.001	<0.01	0.031	<0.01
Muscle hind	<0.001	NA	*0.001	NA
Muscle fore	<0.001	NA	*0.001	NA
Whole blood	0.005	NA	0.029	NA
Plasma	0.004	NA	0.035	NA
Total	NA	97.3	0.035	95.9

NA: Not applicable

†: Urine, faeces, cage wash and milk only.

*: Results calculated from data less than 30 dpm above background.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the test material is rapidly excreted by ruminants, mainly as the unchanged compound. Low doses are excreted mainly via the urine, indicating a high level of absorption. The higher dose showed an increased proportion excreted in the faeces, possibly indicating a lower absorption efficiency. Excretion via the milk was minimal. There was no evidence for accumulation in tissues; levels were negligible in most tissues within 23 hours of the final dose.

Executive summary:

The metabolism and residue profile of the test material was assessed according to EPA Pesticide Assessment Guidelines, Subdivision 0, Series 171-4: Nature of the Residue in Livestock (Residue Test Guidelines, OPPTS 860.1300, Nature of the Residue - Plants, Livestock, US Environment Protection Agency, August 1996), and FAO Guidelines as Recommended by EU Commission Directive 96/68/EC Annex 1, Section 6.2, (21 October 1996) and in compliance with GLP.

This report describes a study in which the metabolism and residue profile of the test material, an established broad leaf herbicide, was investigated in the lactating goat. The test substance was uniformly radiolabelled with carbon-14 in the aromatic ring. 

Two lactating goats each received a twice-daily oral administration of [14C]-test material in gelatin capsules over a period of 7 consecutive days. Goats 1 and 2 received doses at nominal levels of 5 ppm and 50 ppm of daily food consumption per day, respectively. Milk was collected twice daily immediately prior to each dosing. Faeces and urine were collected during the day prior to the first dose and at 24 h intervals after the first dose. The animals were killed at ca.23 h after the last dose and selected tissues collected. All biological samples were assayed for total radioactivity by liquid scintillation counting (LSC), either directly or following sample combustion.

The major route of excretion at both dose levels was via urine, with 81.0 % (Goat 1, low dose) and 64.5 % (Goat 2, high dose) of the administered dose recovered.

Excretion in faeces accounted for 11.0 % and 24.9 % of the administered dose for the low dose and high dose levels, respectively. Radio-HPLC analysis of urine and faeces from Goat 2 ndicated that the test material accounted for a mean of ca. 94.1 % of the radioactivity present in urine and a mean of ca.92.5 % of the radioactivity present in faeces. This indicates that ca.83.7 % of an oral dose of [14C]-test material was excreted as parent molecule at the high dose level. Radio-HPLC analysis of urine and faeces from Goat 1 indicated that the test material accounted for a mean of ca. 96.4 % of the radioactivity present in urine and a mean of ca. 90.3 % of the radioactivity present in faeces. This indicates that ca. 88.0 % of an oral dose of [14C]-test material was excreted as parent molecule at the low dose level.

Concentrations of radioactivity in milk were low at both dose levels. At the 50 ppm dose level a maximum concentration of 0.013 μg equiv/g at 128 and 152 h post dose declined to 0.007 μg equiv/g at 176 h post dose. Levels did not rise above 0.001 μg equiv/g at the low dose level. For both dose levels, the total levels of residues in milk accounted for 0.02 % of the administered dose.

The highest tissue residue levels were found in the kidney where the concentrations were 0.007 μg equiv/g (low dose) and 0.097 μg equiv/g (high dose). Residue levels in the liver were 0.001 μg equiv/g (low dose) and 0.031 μg equiv/g (high dose). The overall distribution of total radioactivity within the tissues was broadly similar for both dose levels, with very low residue levels found in the renal fat, omental fat, hind muscle and fore muscle. Concentrations of total radioactivity in the whole blood and plasma were also low with levels of only 0.005 μg equiv/g and 0.004 μg equiv/g, respectively, recorded for the low dose goat. Corresponding levels for the high dose goat were 0.029 μg equiv/g and 0.035 μg equiv/g.

The amount of total radioactivity recorded in tissues of Goat 1 and Goat 2 represented <0.01 % of the administered dose.

The overall recovery of the total administered radioactivity was high for both Goat 1 (97.3 %) and Goat 2 (95.9 %).

Sub-samples of Goat 1 urine (48 h and 168 h) were concentrated under nitrogen to a lower volume. Sub-samples of Goat 2 urine (48 h and 168 h) were centrifuged only.

Faeces samples (48 h and 168 h) from Goat 1 and Goat 2 were extracted with methanol. The radioactivity in the combined methanol extracts was then quantified by liquid scintillation counting (LSC). For faeces and tissues, radioactivity remaining in the post-extracted solids (PES) was quantified by combustion analysis and LSC. Goat 1 faeces combined methanol extracts were then partitioned with hexane. For each sample, the solvent extracts were combined and concentrated under nitrogen to a lower volume.

Sub-samples of milk (56 h and 152 h) were processed using acetonitrile precipitation to remove protein followed by diethylether and hexane partitioning to remove fats. The aqueous phase was concentrated, combined with acetonitrile washes of the protein pellet and concentrated further.

No procedural losses occurred following processing of Goat 1& (48 h and 168 h) urine samples. Procedural recovery values of 99.8 % and 97.2 % for the 48 h and 168 h urine samples from Goat 2, respectively, indicated quantitative recovery of residues.

Faeces final extraction efficiency values of 95.1 % TRR (Total Radioactive Residue) (48 h) and 87.3 % TRR (168 h) were obtained for Goat 1, with values of 97.0 % TRR and 94.6 % TRR observed for Goat 2 48 h and 168 h faeces samples, respectively.

Final extraction efficiencies of 29.6 % and 30.8 % TRR were observed for the 56 h and 152 h milk samples, respectively. The low values for milk were due to the low initial residue levels of 0.011 μg equiv/g to 0.013 μg equiv/g and attributed to the low levels of radioactivity in the concentrated initial extracts. No or minor procedural losses occurred following hexane partitioning. HPLC analysis of Goat 2 milk (56 h and 156 h) showed the presence of the test material by co-chromatography, but at levels close to or less than the limit of quantification.

Liver and kidney samples from Goat 1 and Goat 2 were processed using a sequential extraction approach due to low initial extraction efficiencies using solvent alone. Aqueous methanol extraction was followed with protease enzyme extraction, pepsin enzyme extraction and finally by caustic methanol extraction. Initial extraction efficiencies with methanol, followed by methanol/water (9:1, v/v), were highest in the liver with the combined initial extracts representing 58.0 % TRR (0.018 ppm). Kidney combined initial extracts represented 47.7 % TRR (0.046 ppm). Minor losses of radioactivity were observed following processing, such that the final liver and kidney concentrated aqueous/methanol extracts represented 54.7 % TRR (0.017 ppm) and 44.3 % TRR (0.043 ppm), respectively.

Residues remaining in post extracted solids from both the liver and kidney samples were further extracted by protease enzyme hydrolysis, recovering 5.7 % TRR (0.002 ppm) and 3.5 % TRR (0.003 ppm), respectively. Following pepsin enzyme hydrolysis a further 20.1 % TRR (0.006 ppm) and 20.2 % TRR (0.020 ppm) was recovered from the liver and kidneys, respectively. Similar levels of radioactivity were released from the liver and kidney PES samples following caustic extraction, where up to 2.7 % (0.002 ppm) and 20.5 % TRR (0.013 ppm) was released following incubations at 58 °C for 20 h and 70 °C for 48 h, respectively.

Processed urine from both goats was analysed directly by reverse phase HPLC with on-line radiodetection with peak integration. Concentrated and processed initial extracts of faeces, liver, kidneys and milk were analysed by reverse phase HPLC with fraction collection/LSC. Further extracts were not processed as the small quantity of radioactivity present precluded meaningful attempts to identify the species present.

Radio-HPLC analysis of urine samples from both the low and high level dosed goats indicated that the test material was the major residue accounting for 96.9 % TRR (10.33 % dose, this refers to the cumulative dose over the 7 days of the study) and 95.9 % TRR (13.14 % dose) in Goat 1&, 48 h and 168 h, respectively. Totals of 95.4 % TRR (17.42 % dose) and 92.7 % TRR (8.79 % dose) were recovered in the 48 h and 168 h urine samples, respectively, from Goat 2&. Three additional minor components were detected in Goat 1& urine, accounting for up to 0.16 % dose (48 h) and 0.21 % dose (168 h). In Goat 2& urine six additional minor components were detected, accounting for up to 0.28 % dose in the 48 h sample and 0.13 % dose in the 168 h sample. These minor components were not further identified due to the small amount of radioactivity present.

In the faeces samples, the test material was also the major component detected at both dose levels. Parent accounted for 2.38 % dose (93.9 % TRR) in the 48 h sample and 3.66 % dose (91.1 % TRR) in the 168 h sample, at the high dose level. Minor (unidentified) components accounted for up to 0.06 % dose in the 48 h sample and 0.09 % dose (2.2 % TRR) in the 168 h sample. At the low dose level parent accounted for 2.19 % dose (93.3 % TRR) in the 48 h sample and 1.14 % dose (87.3 % TRR) in the 168 h sample. A single minor unknown component, representing 0.04 % dose (1.8 % TRR) was detected in the 48 h sample.

Due to the very low residue levels (0.003 ppm, 30.8 % TRR) in the 56 h milk extracts from Goat 2, no radiolabelled residues were detected during HPLC analysis. Analysis of the 152 h milk sample indicated the presence of a single unidentified component (29.6 % TRR, 0.004 ppm). There was evidence for the presence of the test material in this sample, but at levels less than the limit of quantification. Confirmation of the test material in Goat 2 urine (168 h), faeces (168 h) and kidney was carried out using ESI-LC-MS. The reconstructed ion chromatograms for the urine, faeces and kidney were consistent, with each containing the ion at m/z 213 and its chlorine atom associated isotope peak at m/z 215, corresponding to the peak in the radiochromatograms of each sample analysis. The fragment ions at m/z 141 and 143 were also present. The retention time, full scan spectrum and product ion spectrum of this component were consistent with those observed for authentic the test material in each sample type, thereby confirming the component as the test material.

HPLC analysis of the liver aqueous/methanol extracts, containing 0.017 ppm (54.7 % TRR), showed the presence of 1 polar unknown component at a retention time of 5 min. HPLC analysis of the kidney aqueous/methanol extracts, containing 0.043 ppm (44.3 % TRR), showed the presence of 3 components. The most abundant of these represented 0.030 ppm (31.1 % TRR) and was identified as the test material by co-chromatography. The remaining 2 unidentified polar components represented 0.003 ppm and 0.010 ppm (2.7 % TRR and 10.5 % TRR). The kidney pepsin enzyme hydrolysis extract contained 1 component at 0.016 ppm (16.6 % TRR), corresponding to the test material by chromatography. 

It is concluded that under the conditions of the study the test material is rapidly excreted by ruminants, mainly as the unchanged compound. Low doses are excreted mainly via the urine, indicating a high level of absorption. The higher dose showed an increased proportion excreted in the faeces, possibly indicating a lower absorption efficiency. Excretion via the milk was minimal. There was no evidence for accumulation in tissues; levels were negligible in most tissues within 23 hours of the final dose.