Mecoprop

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1977 to March 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to investigate the absorption, distribution and excretion of radioactivity following either single or repeated oral administration of the test material to the rat.

GLP compliance:

no

Remarks:

Study pre-dates GLP.

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Main study: 160 - 210 g; Bile duct cannulation study: 3 males 370 - 400 g.
Single dose tissue distribution study: 160 - 190 g.
Repeated dose tissue distribution study (up to 20 doses): 160 - 200 g.
Repeated dose tissue distribution study (up to 40 doses): 160 - 180 g.
- Housing: The animals were housed four per cage in solid floor polypropylene cages until transfer to all-glass metabolism cages or wire mesh bottom cages for the plasma and tissue distribution studies.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum of four days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Photoperiod: Natural lighting conditions
- Fasting period: Prior to dosing, all animals were fasted for 17 hours, but had free access to mains tap water.

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

Distilled water containing 1 % (w/v) methyl cellulose

Details on exposure:

DOSE PREPARATION
- The stated quantities of labelled and unlabelled test material were ground together using a glass pestle and mortar until the mixture reached the consistency of a fine powder. A suitable volume of distilled water containing 1 % (w/v) methyl cellulose was added slowly with continuous grinding. The suspension was transferred to a measuring cylinder with repeated washing until the required volume was indicated.
- The resultant suspension was then dispensed into portions for each of the individual studies and stored at -18 °C until required.
- The test material for the whole body autoradiography and repeat dose studies was made up immediately before use.
- Before dosing, the material was stirred for at least 30 minutes using a magnetic stirrer. Immediately before dosing, portions were sampled and assayed for radioactivity to confirm homogeneity.

Tissue distribution study: Rats were each given a dose of 11.6 μCi per 200 g in a volume of 1.0 mL per 200 g.
Bile duct cannulation study: Each rat received an oral dose of the 14C-test material at a dose level of 107 mg/kg bodyweight.
Whole body autoradiography: Approximately 50 μCi of 14C-test material at a dose level of 107 mg/kg bodyweight.
Repeated dose tissue distribution study (up to 20 daily doses): 1 mg/kg bodyweight in a volume of 1 mL/200 g and were given 3.78 μCi/200 g of bodyweight daily.
Repeated dose tissue distribution study (up to 40 daily doses): Dose level of 1 mg/kg bodyweight in a volume of 0.5 mL/200g.

Duration and frequency of treatment / exposure:

Single dose study: Each animal was dosed once.

Repeat dose study:
- Tissue distribution study 1: Daily oral dose for up to 20 days.
- Tissue distribution study 2: Daily oral dose for up to 40 days.

Dose / conc.:

1 other: mL/ 200g bw

Remarks:

Single dose study: Excretion study

Dose / conc.:

11.6 other: μCi per 200 g

Remarks:

Single dose study: Tissue distribution study

Dose / conc.:

107 mg/kg bw/day (nominal)

Remarks:

Single dose: Bile duct cannulation study

Dose / conc.:

107 mg/kg bw/day (nominal)

Remarks:

Single dose study: Whole body autoradiography

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Repeat dose study: Tissue distribution studies

No. of animals per sex per dose / concentration:

Single dose study:
- Excretion study: 3/sex/dose
- Tissue distribution study: 14/sex/dose
- Bile duct cannulation study: 3 males
- Whole body autoradiography: 3/sex/dose

Repeat dose study:
- Tissue distribution study 1: 10/sex/dose
- Tissue distribution study 2: 14 male rats

Control animals:

no

Details on dosing and sampling:

Each animal was dosed by oral gavage with an appropriate volume, dictated by its body weight, so that it received 107 mg of (14C)-test material/kg body weight. After dosing, portions of the material were again sampled to confirm homogeneity. The suspension was stirred continuously during dosing.

SINGLE DOSE STUDY
Excretion Study:
- Tissues and body fluids sampled: Faeces, urine, expired air.
- Time and frequency of sampling: Faecal collections were made a 24 hour time intervals and urine collections 6, 12, 24, 48, 72, 96, 120, 144, 168 and 192 hours after treatment. For the first 24 hours the expired air was monitored in two animals (one male and one female) to determine the release of 14CO2.
- Other: Dry, carbon dioxide-free air, was drawn through the metabolism cages and then through two scrubbing towers each containing 4N sodium hydroxide solution (100 mL), the contents of which were changed at 12 hr intervals. The flow rate was maintained between 0.25 and 0.4 litres/minute. At the end of the study each cage was washed internally with water (approximately 30 mL) and the washing retained for assay.
Portions of each urine sample (100 μL) and each scrubbing solution (500 μL) were assayed for radioactivity by direct liquid scintillation counting. Cage washings, which combined particulate material, and faeces, were homogenised (the faeces being diluted 1:4 (w/v) with saline) and portions of each homogenate were combusted prior to assay.

Tissue Distribution Study:
- Tissues and body fluids sampled: Fat, adrenals, brain, colon (minus contents), eyes, gonads, heart, ileum (minus contents), kidney, liver, lung, residual carcass, skeletal muscle, skin, spleen, stomach (minus contents) and thyroid.
- Time and frequency of sampling: At specified intervals, (3, 6, 24, 48, 96, 144 and 192 hours after dosing) groups of four animals (two males and two females) were killed and both blood and selected tissues removed.
Other:
- Plasma was prepared by centrifugation of each blood sample and portions (100 μL) solubilised in a counting vial containing Soluene 350 (200 μL). Overnight incubation at 37 °C was routinely employed for all Soluene digestions.
- Following removal, each tissue was cleaned, where appropriate, from adhering connective tissue or fat, weighed and stored at -20 °C prior to analysis.
Minced tissue samples were homogenised using a Silverson mixer/homogeniser fitted with 5/8 inch microhead, in 4 volumes of cold physiological saline. Each residual carcass was minced whilst frozen using a stainless steel Hobart food cutter with a mincer attachment. The mince was then homogenised in 4 volumes of saline using a Silverson mixer fitted with a standard head. Portions of each homogenate (200 µL) were either digested in Soluene or, if highly coloured, were combusted to (14C)-CO2 prior to being counted.

Bile Duct Cannulation Study:
Rats were anaesthetised in an atmosphere of 1.5 % (v/v) Halothane in oxygen using a Fluotec Mark II Vapouriser. A small glass vessel (volume approx. 5 mL) was then asceptically implanted into the peritoneum. The glass vessel had two tube connections, one of which was attached to the common bile duct via a small plastic cannula which allowed the bile to empty into the vessel. The other tube was exteriorised through the mid lumbar region adjacent to the spine and permitted free sampling of the collected bile, A small piece of rubber pressure tubing was fitted over the exteriorised outlet to secure it. Internal abdominal musculature was sutured with surgical thread and the skin closed with sterile autoclips. Each rat was allowed to recover for 24 hr after which it was fasted for 12 hr. By this procedure the rats were able to move freely and yet allowed free sampling of the collected bile. At specified intervals over the next 24 h the bile was sampled via a syringe attached to a short length of cannula.
- Tissues and body fluids sampled: Bile.
- Time and frequency of sampling: The sampling intervals were 1, 2, 4, 12 and 24 hr after dosing.
- Other: At the end of the 24 hour period the rats were killed and the blood and following tissues obtained for subsequent analysis: Liver, kidney, fat, heart, lung, spleen, stomach (minus contents), ileum (minus contents), colon (minus contents), skin and muscle.
Portions of each bile sample (100 μL) were added to liquidscintillator (15 μL) and counted directly. The 0 - 24 h urine and faeces were also assayed for radioactivity.

Whole Body Autoradiography:
- Tissues and body fluids sampled: Autoradiography of whole body slices was carried out.
- Time and frequency of sampling: Two rats (one male and one female) were killed at each of the three specified time intervals after dosing (6 hr, 24 hr, 48 hr).
Rats were anaesthetised using diethyl ether and frozen by innnersion in a mixture of solid carbon dioxide and acetone. The thoroughly frozen carcass was removed from the freezing mixture after approximately 30 minutes and rubbed with paper tissue to remove acetone. Legs and tail were removed with heavy bone cutters and the hair removed by electric clippers. The animal was then returned to the freezing mixture for a further 30 minutes. To fix the animal in the saw clamping device it was embedded in a mould with cellulose pulp (50 % cellulose fibre in 1 % (w/v) ethanol). The embedded rat was returned to solid carbon dioxide/acetone mixture for 15 minutes followed by immersion in liquid nitrogen for 40 minutes. Each animal was sawn into approximately 2 mm longitudinal slices which were kept frozen. The cellulose packing was removed and the surface of each section scraped to remove debris. Sections produced in this fashion were freeze dried inside a Speedivac freeze-drier and then exposed against Kodirex X-ray film at -30 °C. Each film was subsequently developed in Kodak D19 developer. Exposure times were 36 hr for animals killed 6 hr after treatment, 108 hr for those killed after 24 hr and 144 hr for the 48 hr kill.

REPEAT DOSE STUDY
Tissue Distribution Study (up to 20 daily doses):
- Tissues and body fluids sampled: The animals were exsanguinated during diethyl ether anaesthesia and blood was removed by heart puncture and placed in heparinised plastic tubes. Part or all of the following tissues were removed, cleaned of adhering tissue and fat and placed in pre-weighed containers: Liver, kidneys, skeletal muscle, fat, adrenals and thyroids. The blood was centrifuged and the plasma separated. The concentration of radioactivity in plasma was determined by direct liquid scintillation counting. The tissues were treated as in the single dose study.
- Time and frequency of sampling: Two male and two female rats were killed 24 hours after the 1st, 10th, 15th and 20th dose and also 96 hours after the 20th dose.
- Other: The following group of tissues was removed and stored at -20 °C in case subsequent analysis was required: Heart, lungs, spleen, gonads, stomach (minus contents), ileum (minus contents), colon (minus contents), brain, eyes and skin.

Tissue Distribution Study (up to 40 daily doses):
- Tissues and body fluids sampled: Same as tissue distribution study (up to 20 daily doses).
- Time and frequency of sampling: Two animals were killed after 21, 26, 29, 33, 37, 41 and 44 days and the same tissues were taken and processed.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine samples containing the highest concentrations of radioactivity. They were stored at -20 °C prior to use.
- Time and frequency of sampling: Not specified.
- From how many animals: 3 males and 2 females.
- Method type(s) for identification: TLC, radiochromatograms (LSC)

ANALYTICAL METHOD: Thin layer chromatography
- Portions (50 μL) of each urine extract were submitted to thin layer chromatography using 20 x 20 cm glass plates precoated with Silica Gel 60, (layer thickness 0.25 mm) containing a fluorescent indicator, F254. The plates were developed in a mixture (100 mL) of chloroform cyclohexane: acetic acid (80: 20: 10 (v/v)). Authentic test material dissolved in ethanol was submitted to the same chromatographic techniques for comparison.

Solvent Extraction of Urine
- The pH of each urine sample was adjusted to 4.0 using 0.1 M hydrochloric acid. Samples (0.5 mL) were extracted for 10 minutes using ethyl acetate (3 x 5 mL) using a rotary mixer, and then centrifuged. The pooled organic phase was evaporated to dryness under a stream of nitrogen. The residue was redissolved in ethyl acetate (0.5 mL) and samples (50 or 100 μL) submitted to thin layer chromatography.

Hydrolysis with Acid and Alkali
- Each urine sample was hydrolysed with both acid and alkali. The sample (0.5 mL) was heated to boiling point in a Kjeldahl flask in the presence of 2.5 M sulphuric acid (2 mL) or 5 M
sodium hydroxide solution (2 mL) for two hours. The solutions were cooled, adjusted to pH 4 and extracted with ethyl acetate (3 x 5 mL) for 10 minutes on a rotary mixer. The pooled solvent extract was evaporated to dryness under a stream of nitrogen. The residue was redissolved in ethyl acetate (0.1 mL) and samples (10 μL) submitted to thin layer chromatography.

Enzyme Hydrolysis using β-glucuronidase
- The pH of the following urines was adjusted to 5.5 with acid: Rat 1 (male), 12 - 24 hours; Rat 2 (male, 12 - 24 hours; Rat 1 (female, 24 - 48 hours and Rat 2 (female), 12 - 24 hours. A sample (0.5 mL) of this urine was then incubated for 15 hr at 37 °C in the presence of approximately 10 000 units of β-glucuronidase dissolved in acetate buffer at pH 5.5. The enzyme was obtained from Helix pomatia and had some aryl sulphatase activity. A control incubation of each urine sample was carried out using the same conditions except the enzyme was omitted. Following incubation all samples were adjusted to pH 4 and extracted using ethyl acetate and submitted to thin layer chromatography.

ANALYTICAL METHOD
Preparation of radiochromatograms
- Radioactivity on the thin layer plates was located using a Radiochromatogram Spark Chamber and recorded on Polaroid film. Areas of the adsorbent layer shown to contain radioactivity were scraped off and added to scintillator (15 mL) containing 4.0 % (w/v) Cab-o-Sil, shaken thoroughly, and counted in a liquid scintillation spectrometer.
- Autoradiography was used to complement the radiochromatograms and to indicate any areas of low radioactive content not previously located.

DETERMINATION OF RADIOACTIVITY
- Sample radioactivity was measured using a Packard Tri-Carb Model 3375 Liquid Scintillation Spectrometer (Packard Instrument Ltd.) with optimised gain and window settings. Aliquots of radioactive solutions were counted in a scintillant consisting of 8 % (w/v) naphthalene, 0.5 % (w/v) PPO (2,5-diphenyloxazole) and 0.005 % (w/v) dimethyl POPOP (1,4,-di-(2-(4-methyl-5-phenyloxazolyl)benzene) in xylene: 1,4 dioxane: 70 % (v/v) methanol in ethanol (1:1:1 (v/v)).
- The degree of quenching was determined by use of an automatic external standard. Aqueous samples were added directly to scintillator (15 mL). Soluene digests were also diluted with the same volume of scintillator. Other homogenates were combusted using a Sample Oxidiser Model 305 and the 14 C-carbon dioxide absorbed in ethanolamine (7 mL) which was mixed with a scintillation cocktail containing 1. 5 % (w/v) PPO and 0.1 % (w/v) bis-MSB in toluene (3 mL) and methanol (9 mL). All samples were shaken thoroughly and counted for at least 10 minutes or until 10 000 counts bad accumulated.
- Soluene digestion resulted in a degree of quenching but the quench curve was identical to the normal quench curve and corrected data could therefore be calculated using a conventional programme. Soluene containing 'blanks' gave similar counts to those obtained using xylene based scintillator alone.
- 14C-labelled standards were routinely combusted using the Sample Oxidiser. Combustion eddiciency was in excess of 95 %. All reported combustion data on tissues are therefore uncorrected.

CALCULATION OF RESULTS
- The results are expressed to three experimentally valid figures, and include a consideration of the minimum limits of detection. No statistical analysis of results was conducted because of the small number of animals used.

Type:

absorption

Results:

Rate and extent of absorption: Rapid, > 90 % based on urinary and biliary excretion.

Type:

distribution

Results:

Widely distributed.

Type:

other: Accumulation

Results:

Potential for accumulation: No potential for accumulation, highest residues in fat after 8 days.

Type:

excretion

Results:

Rate and extent of excretion: Rapid, approximately 90 % within 48 hours, mainly via urine (81 %).

Type:

metabolism

Results:

Metabolism in animals: Limited, about 45 % excreted as parent. Main metabolic step: Hydroxylation.

Details on absorption:

The mean cumulative percent excretion of the radioactive dose in urine and faeces illustrates
that the pattern of elimination was similar in both male (total = 90.0 %) and female rats (total = 96.0 %). It also demonstrates that orally administered test material was well absorbed and rapidly excreted in the rat.

Details on distribution in tissues:

SINGLE DOSE STUDY
- High concentrations of plasma radioactivity were present in both sexes within six hours of test material administration. The peak plasma concentration in female rats occurred three hours after dosing (mean = 326.00 μg equivalents/mL), whereas in male rats it occurred six hours after dosing (mean = 319.00 μg equivalents/mL). With the exception of the latter plasma concentration, the levels of radioactivity in female rats were consistently higher than those in males throughout the 192 hr period of the study.
- The decline in the concentrations of plasma radioactivity occurred more rapidly in males than in females. After peaking at 6 hr in the male rat (mean = 319.00 μg equivalents/mL) the level was markedly reduced by 24 hr (mean = 46.50 μg equivalents/mL), but in the female rat the 24 hr concentration (mean = 130.00 μg equivalents/mL) remained relatively high in comparison to that of the 6 hr sample (mean = 304.00 μg equivalents/mL). Forty-eight hours after test material administration, the plasma concentration of radioactivity was very low in both sexes (mean = 6.02 μg equivalents/mL).
- The tissue distribution study showed that, with the exception of the brain (mean sample at 3 hr = 11.8 μg equivalents/g), all the organs studied contained high levels of radioactivity at the early sample times (3 and 6 hr). Typical mean values found 3 hr after test material administration were 186.00 μg equivalents/g of liver, 126.00 μg equivalents/g of kidneys and 118.00 μg equivalents/g of thyroid gland. The high concentrations of radioactivity in the stomach peaked at 3 hours in males (mean = 119.00 μg equivalents/g) and 6 hours in females (mean = 123.00 μg equivalents/g).
- The only sex difference found in the tissue distribution study concerned the gonads. The ovaries consistently contained higher concentrations of radioactivity than did the testes. The highest mean levels found in the ovaries and testes were 88.60 and 47.80 μg equivalents/g 3 and 6 hr after dosing respectively.
- In general there was a steady decline in the tissue concentrations of radioactivity, such that 192 hr after dosing, the levels were very low. There were, however, a number of exceptions. In the fat of both sexes, the decline in the radioactivity concentration was very gradual. The maximum mean level of 59.40 μg equivalents/g was reached 6 hr after test material administration, thereafter it declined slowly and by 192 hr, the mean concentration was 22.20 μg equivalents/g. The pattern was similar in the skin though the decline from the mean 3 hr peak concentration of 72.70 μg equivalents/g to the mean 192 hour concentration of 8.18 μg equivalents/g was more rapid.
- The rate of absorption of the test material was high in both sexes but it appeared to be more quickly absorbed in the females from the evidence of relatively high initial plasma radioactivity concentrations. However, the clearance of plasma radioactivity occurred more rapidly in the male rats.

Whole Body Autoradiography
- The whole body autoradiographs of both male and female rats were in good agreement with the pattern of results found in the tissue distribution study. At 6 hr after treatment, all the major organs such as stomach, liver and kidneys contained high concentrations of radioactivity (Plates 1 and 2). The notable exception was the brain and spinal cord. There was a fairly even distribution of radioactivity throughout the gastro-intestinal tract.
- Twenty-four hours after test material administration, the concentrations of radioactivity were still relatively high in both male and female rats (Plates 3 and 4), but particularly in the kidneys and gastrointestinal tract. Rats of both sexes had similar concentrations of radioactivity after 48 hr, with the exception of the female kidneys which were noticeably high (Plate 5). There were still detectable levels of radioactivity present in skin and/or subcutaneous fat 48 hr after dosing.

REPEAT DOSE STUDY
Tissue Distribution Study (up to 20 daily doses)
- The results demonstrate that continuous daily administration of the test material for up to 20 days progressively increased tissue concentrations of radioactivity in male rats. In some tissues, such as muscle, the increase was negligible (0.02 μg equivalents/g on Day 1 increasing to 0.07 μg equivalents/g on Day 21). In most tissues, however, there was a marked increase in tissue concentrations between Days 1 and 21. Typical mean increases found with liver, kidneys and adrenal glands were 0.08 up to 0.43 μg equivalents/g, 0.29 up to 1.45 μg equivalents/g and 0.06 up to 0.28 μg equivalents/g respectively. Mean plasma concentrations of radioactivity in the male rat increased from 0.08 μg equivalents/mL on Day 1, to 0.48 μg equivalents/mL by Day 21. Four days after administering the final dose of the test material however, the concentration of radioactivity in nearly all these tissues was markedly reduced. The one exception was fat.
- In the male rat, the level of radioactivity in the fat increased to a plateau by Day 16 (mean = 0.54 μg equivalents/g). Twenty four hours after the final dose of the test material (Day 20) the mean concentration was 0.52 μg equivalents/g, but 96 hr. afterwards the mean concentration was reduced to only 0.42 μg equivalents/g.
- The disposition of radioactivity in the female rat differed from that of the male in that peak tissue concentrations were generally attained by Day 11 (e.g. kidney mean = 1.28 μg equivalents/g, the values thereafter tended to decline. Although the male tissues demonstrated the progressive increase in tissue concentrations, those of the females tended to be higher at the early sample times.

Tissue Distribution Study (up to 40 daily doses)
- In view of the accumulation of radioactivity in male rat tissues from the 20 day repeat dose study, a 40 day study was performed. It was found that there was no increase in tissue concentrations of radioactivity between Days 21 and 41. The levels in certain tissues, such as the liver, remained very consistent throughout this period (mean ±SD = 0.20 ± 0.02 μg equivalents/g). The highest concentrations of radioactivity were found in the kidneys (mean ±SD = 0.56 ± 0.10 μg equivalents/g). With the exception of fat, concentrations of radioactivity in the tissues 96 hr after the 40th dose were all markedly reduced.

Details on excretion:

Single Dose Study
- There was no radioactivity detected in either of the scrubbing towers used for absorbing carbon dioxide from the selected animals.
- The major proportion of the radioactive dose was excreted in the urine of both male (mean = 81.8 %) and female rats (mean = 89.2 %). The majority of the radioactivity was found in the 0-24 hr urine (mean = 61.3 %), though the rate of elimination was more rapid in the males (2.9 %/hr) than in females (2.3 %/hr) during this period.
- Faecal excretion of radioactivity was a minor route of elimination. It accounted for a mean of 8.2 and 6.8 % of the radioactive dose administered to male and female rats respectively. Most of this radioactivity was recovered in the 0-24 hr samples (mean = 4.9 %) but low quantities were excreted between 24 and 192 hr post dosing (mean = 2.7 %).
- The cage washings collected at 192 hr contained 0.1 % of the administered radioactivity in both sexes.
- The mean cumulative percent excretion of the radioactive dose in urine and faeces illustrates that the pattern of elimination was similar in both male (total = 90.0 %) and female rats (total = 96.0 %).

Bile Duct Cannulation Study
- The rate of bile production in all three rats was of the same order with mean values of 0.8, 0.7 and 0.9 ml/hr in Rats 1 (male), 2 (male) and 3 (male) respectively. The mean percent of the radioactive dose excreted in the 0 - 24 hour bile was 64.8 %. The majority of this radioactivity was collected in the 4 - 24 hr sample (mean = 54.6 %) although the rate of excretion in each animal was generally steady over the whole experimental period. The 0 - 24 hr urine contained between 9.4 and 18.2 % of the radioactive dose (mean = 13.9 %), and the 0 - 24 hr faeces contained between 0.1 and 0.4 % (mean= 0.3 %). The mean recovery over the 0 - 24 hr period in bile, urine and faeces was 79.1 %.
- The plasma and tissue concentrations of radioactivity were generally higher than those in the tissue distribution single dose study and this may reflect the comparatively poor condition of the bile duct cannulated animals following surgery. As examples, the mean concentrations of radioactivity in the liver, kidneys and plasma 24 hr after dosing in this study were 52.40, 59.40 and 112.10 μg equivalents/g or mL respectively, whereas the corresponding values in the tissue distribution study were 20.90, 25.10 and 46.50 μg equivalents/g or mL.

Metabolites identified:

yes

Remarks:

Unknown metabolite with RF value of 0.12

Details on metabolites:

Metabolism Study
Thin layer chromatography was used to assess the radiochemical purity of the (14C)-test material. It was confirmed that the test article was at least 97.3 % radiochemically pure.
Solvent extraction of urine Between 98.0 and 99.1 % of the radioactivity present in rat urine was extracted with ethyl acetate. The extracts were concentrated and submitted to thin layer chromatography. Three major radioactive fractions were resolved and these were submitted to liquid scintillation counting.
In the four rat urines extracted, the fraction corresponding in Rf to that of (14C)-test material (Rf = 0.66) accounted for a mean of 44.9 % of the recovered radioactivity. Another major fraction, which remained at the origin of the plate, accounted for a mean of 39.2 %. The third fraction had an Rf of 0.12 and represented a mean of 11.7 % of the recovered radioactivity.

The ratio of these three radioactive fractions was markedly altered following either acid or alkaline hydrolysis. The component corresponding in RF to the test material accounted for a mean of 78.5 and 88.6 % of the recovered radioactivity after acid and alkaline hydrolysis respectively, whereas the same values for the fraction which was retained at the origin were 11.5 and 1.4 %. The component with an RF of 0.12 represented a mean of 7.0 and 5.8 % after acid and alkaline hydrolysis.

Incubation with β-glucuronidase resulted in an increase in the fraction, corresponding in RF to the test material, to a mean of 82.4 % but at the expense of the polar fraction retained at the origin which then only represented a mean of 3.8 % of the recovered radioactivity. However a similar change in ratio was observed following incubation in the absence of the enzyme, though the corresponding values of 70.6 and 15. 6 % indicated a marginally lower conversion of one to the other. Nevertheless it was clear that the polar fraction was very labile and could break down liberating the component corresponding in RF to the test material. The fraction with an RF of 0.12 was unchanged following incubation with β-glucuronidase.

Autoradiography of a thin layer plate previously examined in the spark chamber demonstrated good correlation between the two systems of examination. However the more sensitive autoradiographic technique showed the presence of a number of minor radioactive components hitherto unobserved. These had RF values of 0.04, 0,08, 0.21, 0.34, 0.38 and 0.52. No attempt was made to identify these components.

Conclusions:

Under the conditions of this study, there was a rapid and extensive absorption of radioactivity. It was concluded that an enterohepatic circulation of radioactivity was occurring and that the majority of the radiolabelled material involved was eliminated mostly via the urine (> 90 % in the first 48 hours). The test material was widely distributed. The toxicologically significant compound was the parent in mammals, plant and environment. The metabolism in animals was limited, autoradiography indicated the presence of a metabolite with an RF value of 0.12.

Executive summary:

The metabolic fate of the test material was studied in rats following both single and repeated oral administration.

Following a single oral dose of (14C)-test material at a dose level of 107 mg/kg body weight, there was a rapid and extensive absorption of radioactivity. This was demonstrated by the early peak plasma radioactivity concentrations in both male (mean 6 hr sample = 319.00 µg equivalents/mL) and female (mean 3 hr sample = 326.00 µg equivalents/mL) rats and also the high proportion of the radioactive dose·excreted in 0 - 24 hr urine (mean = 61.3 %).

The concentrations of radioactivity in plasma were, with the exception of the male 6 hr sample, consistently higher in females than in males throughout the 192 hr period of the study. The rate of decline in plasma radioactivity concentrations was more rapid in male (mean 24 hr sample = 46.50 µg equivalents/mL) than female rats (mean 24 hr sample = 130.00 µg equivalents/mL).

The majority of the radioactivity administered was excreted in the urine of both male (mean = 81.8 %) and female (mean = 89.2 %) rats. Nearly all of this was excreted in the first 48 hr after dosing (mean = 81.0 %) with peak elimination occurring during the 0 - 24 hr period (mean = 61.3 %).

Faecal excretion of radioactivity was a minor route of elimination. It accounted for 8.2 and 6.8 % of the radioactivity administered to male and female rats respectively. The majority of this was recovered in the first 24 hr after dosing (mean = 4.9 %). The cage washings collected on completion of the study contained a mean of 0.1 % of the radioactive dose.

No radioactivity was detected in expired air collected during the first 24 hr after a single oral dose of (14C)-test material.

The mean cumulative percent excretion of the radioactive dose in urine, faeces, expired air and cage washings was 90.0 and 96.4 in male and female rats respectively.

Bile duct cannulation studies in three male rats demonstrated extensive biliary excretion of radioactivity. Within 24 hr of a single oral dose of (14C)-test material, a mean of 64.8 % of the radioactive dose was recovered in the bile, the majority of this being excreted between 4 and 24 hr (54.6 %). The 0 - 24 hr urine and faeces contained a mean of 13.9 and 0.3 % of the radioactive dose respectively. From these findings and those of the single dose excretion study, it was concluded that an enterohepatic circulation of radioactivity was occurring and that the majority of the radiolabelled material involved was eventually voided via the urine rather than the faeces.

Tissue concentrations of radioactivity following a single oral dose of (14C)-test material at a dose level of 107 mg/kg body weight were highest at the earliest sample times (3 and 6 hr) but declined substantially in all tissues, except fat, during the 192 hr period of the study. Typical mean values after 3 hr were 186.00 and 126.00 µg equivalents/g respectively for liver and kidneys compared to 0.64 and 1.77 µg equivalents/g after 192 hr. Fat levels remained relatively constant between 24 hr (mean = 39.00 µg equivalents/g) and 96 hr (mean = 38.40 µg equivalents/g) and were still relatively high in comparison to other tissues at 192 hr (mean = 2.2. 20 µg equivalents/g). The skin (mean = 8.18 µg equivalents/g), adrenal glands (mean = 3.77 µg equivalents/g) and ovaries (mean = 6.66 µg equivalents/g) were also relatively high at this time. There was a marked reduction in tissue concentrations of radioactivity between 24 and 48 hr after dosing. The brain concentrations tended to be the lowest throughout the study.

Whole body autoradiography studies indicated a general distribution of radioactivity 6 hr after (14C)-test material administration. The levels in the alimentary tract, with the exception of the female stomach, were particularly high at this time. The autoradiographs complemented the excretion and tissue distribution studies showing a rapid elimination of radioactivity but with a tendency for the skin to retain radioactivity 48 hr after dosing. The kidneys of the female rat also contained a high level of radioactivity at 48 hr, which was in agreement with the tissue distribution study.

The tissue distribution of radioactivity in rats following daily oral administration of (14C)-test material for up to 20 days in females and 40 days in males at a dose level of 1 mg/kg body weight was also studied. The distribution of the radioactivity was similar to that found in the single dose study. There was a progressive increase of radioactivity in all male tissues in the 20 dose study but there was no indication that there was any further increase when the daily dosing regime was extended to 40 days. In general, the tissue concentrations of radioactivity in female rats reached a maximum at Day 11 and thereafter tended to decline. The concentrations of radioactivity were greatest in the kidneys, fat and plasma at all sample times but the levels were generally well below 1 µg equivalent/g of tissue except in a number of kidney samples analysed in the 20 day study. The decline in the levels of radioactivity found in fat after administration of the final dose 14 of (14C)-test material was markedly slower than that shown for other tissues. This was also noted in the single dose study.

A mean of 98.6 % of the radioactivity present in urine was extracted using ethyl acetate. Thin layer chromatography of such extracts revealed three main radioactive fractions. The major of these, accounting for a mean of 44.9 % of the radioactivity recovered from thin layer plates, exhibited the same Rf value as (14C)-test material (0.66). The second most important fraction, representing a mean of 39.2 % of the recovered radioactivity, was very polar and as such was retained at the origin of the thin-layer plates. The third fraction had an Rf value of 0.12 and accounted for 11.7 % of the recovered radioactivity.

Hydrolysis of urine with β-glucuronidase (not free of aryl sulphatase activity), acid or alkali all served to increase the quantity of the fraction corresponding in Rf to (14C)-test material, but correspondingly reduced that of the polar fraction. However this latter component proved to be very labile since extensive breakdown was also shown to occur following incubation in the absence of the enzyme. The fraction with an Rf value of 0.12 was unaffected by these hydrolytic reactions.

These preliminary metabolism studies suggest that a high proportion of (14C)-test material may be absorbed and excreted unchanged. The polar fraction probably represented conjugated (14C)-test material but due to the instability of this fraction, no conclusions as to the nature of the conjugating agent could be drawn.

Autoradiography of thin layer plates indicated the presence, in addition to that of the aforementioned fraction with an Rf value of 0.12, of a number of very minor components with Rf, values of 0.04, 0.08, 0.21, 0.34, 0.38 and 0.52.

Under the conditions of this study, there was a rapid and extensive absorption of radioactivity. It was concluded that an enterohepatic circulation of radioactivity was occurring and that the majority of the radiolabelled material involved was eliminated mostly via the urine (> 90 % in the first 48 hours). The test material was widely distributed. The toxicologically significant compound was the parent in mammals, plant and environment. The metabolism in animals was limited, autoradiography indicated the presence of a metabolite with an RF value of 0.12.

Description of key information

Basic Toxicokinetics In Vivo: Gilbert et al. (1978)

Under the conditions of this study, there was a rapid and extensive absorption of radioactivity. It was concluded that an enterohepatic circulation of radioactivity was occurring and that the majority of the radiolabelled material involved was eliminated mostly via the urine (> 90 % in the first 48 hours). The test material was widely distributed. The toxicologically significant compound was the parent in mammals, plant and environment. The metabolism in animals was limited, autoradiography indicated the presence of a metabolite with an RF value of 0.12.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Additional information

Basic Toxicokinetics In Vivo: Gilbert et al. (1978)

The metabolic fate of the test material was studied in rats following both single and repeated oral administration. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a single oral dose of (14C)-test material at a dose level of 107 mg/kg body weight, there was a rapid and extensive absorption of radioactivity. This was demonstrated by the early peak plasma radioactivity concentrations in both male (mean 6 hr sample = 319.00 µg equivalents/mL) and female (mean 3 hr sample = 326.00 µg equivalents/mL) rats and also the high proportion of the radioactive dose·excreted in 0 - 24 hr urine (mean = 61.3 %).

The concentrations of radioactivity in plasma were, with the exception of the male 6 hr sample, consistently higher in females than in males throughout the 192 hr period of the study. The rate of decline in plasma radioactivity concentrations was more rapid in male (mean 24 hr sample = 46.50 µg equivalents/mL) than female rats (mean 24 hr sample = 130.00 µg equivalents/mL).

The majority of the radioactivity administered was excreted in the urine of both male (mean = 81.8 %) and female (mean = 89.2 %) rats. Nearly all of this was excreted in the first 48 hr after dosing (mean = 81.0 %) with peak elimination occurring during the 0 - 24 hr period (mean = 61.3 %).

Faecal excretion of radioactivity was a minor route of elimination. It accounted for 8.2 and 6.8 % of the radioactivity administered to male and female rats respectively. The majority of this was recovered in the first 24 hr after dosing (mean = 4.9 %). The cage washings collected on completion of the study contained a mean of 0.1 % of the radioactive dose.

No radioactivity was detected in expired air collected during the first 24 hr after a single oral dose of (14C)-test material.

The mean cumulative percent excretion of the radioactive dose in urine, faeces, expired air and cage washings was 90.0 and 96.4 in male and female rats respectively.

Bile duct cannulation studies in three male rats demonstrated extensive biliary excretion of radioactivity. Within 24 hr of a single oral dose of (14C)-test material, a mean of 64.8 % of the radioactive dose was recovered in the bile, the majority of this being excreted between 4 and 24 hr (54.6 %). The 0 - 24 hr urine and faeces contained a mean of 13.9 and 0.3 % of the radioactive dose respectively. From these findings and those of the single dose excretion study, it was concluded that an enterohepatic circulation of radioactivity was occurring and that the majority of the radiolabelled material involved was eventually voided via the urine rather than the faeces.

Tissue concentrations of radioactivity following a single oral dose of (14C)-test material at a dose level of 107 mg/kg body weight were highest at the earliest sample times (3 and 6 hr) but declined substantially in all tissues, except fat, during the 192 hr period of the study. Typical mean values after 3 hr were 186.00 and 126.00 µg equivalents/g respectively for liver and kidneys compared to 0.64 and 1.77 µg equivalents/g after 192 hr. Fat levels remained relatively constant between 24 hr (mean = 39.00 µg equivalents/g) and 96 hr (mean = 38.40 µg equivalents/g) and were still relatively high in comparison to other tissues at 192 hr (mean = 2.2. 20 µg equivalents/g). The skin (mean = 8.18 µg equivalents/g), adrenal glands (mean = 3.77 µg equivalents/g) and ovaries (mean = 6.66 µg equivalents/g) were also relatively high at this time. There was a marked reduction in tissue concentrations of radioactivity between 24 and 48 hr after dosing. The brain concentrations tended to be the lowest throughout the study.

Whole body autoradiography studies indicated a general distribution of radioactivity 6 hr after (14C)-test material administration. The levels in the alimentary tract, with the exception of the female stomach, were particularly high at this time. The autoradiographs complemented the excretion and tissue distribution studies showing a rapid elimination of radioactivity but with a tendency for the skin to retain radioactivity 48 hr after dosing. The kidneys of the female rat also contained a high level of radioactivity at 48 hr, which was in agreement with the tissue distribution study.

The tissue distribution of radioactivity in rats following daily oral administration of (14C)-test material for up to 20 days in females and 40 days in males at a dose level of 1 mg/kg body weight was also studied. The distribution of the radioactivity was similar to that found in the single dose study. There was a progressive increase of radioactivity in all male tissues in the 20 dose study but there was no indication that there was any further increase when the daily dosing regime was extended to 40 days. In general, the tissue concentrations of radioactivity in female rats reached a maximum at Day 11 and thereafter tended to decline. The concentrations of radioactivity were greatest in the kidneys, fat and plasma at all sample times but the levels were generally well below 1 µg equivalent/g of tissue except in a number of kidney samples analysed in the 20 day study. The decline in the levels of radioactivity found in fat after administration of the final dose 14 of (14C)-test material was markedly slower than that shown for other tissues. This was also noted in the single dose study.

A mean of 98.6 % of the radioactivity present in urine was extracted using ethyl acetate. Thin layer chromatography of such extracts revealed three main radioactive fractions. The major of these, accounting for a mean of 44.9 % of the radioactivity recovered from thin layer plates, exhibited the same Rf value as (14C)-test material (0.66). The second most important fraction, representing a mean of 39.2 % of the recovered radioactivity, was very polar and as such was retained at the origin of the thin-layer plates. The third fraction had an Rf value of 0.12 and accounted for 11.7 % of the recovered radioactivity.

Hydrolysis of urine with β-glucuronidase (not free of aryl sulphatase activity), acid or alkali all served to increase the quantity of the fraction corresponding in Rf to (14C)-test material, but correspondingly reduced that of the polar fraction. However this latter component proved to be very labile since extensive breakdown was also shown to occur following incubation in the absence of the enzyme. The fraction with an Rf value of 0.12 was unaffected by these hydrolytic reactions.

These preliminary metabolism studies suggest that a high proportion of (14C)-test material may be absorbed and excreted unchanged. The polar fraction probably represented conjugated (14C)-test material but due to the instability of this fraction, no conclusions as to the nature of the conjugating agent could be drawn.

Autoradiography of thin layer plates indicated the presence, in addition to that of the aforementioned fraction with an Rf value of 0.12, of a number of very minor components with Rf, values of 0.04, 0.08, 0.21, 0.34, 0.38 and 0.52.

Under the conditions of this study, there was a rapid and extensive absorption of radioactivity. It was concluded that an enterohepatic circulation of radioactivity was occurring and that the majority of the radiolabelled material involved was eliminated mostly via the urine (> 90 % in the first 48 hours). The test material was widely distributed. The toxicologically significant compound was the parent in mammals, plant and environment. The metabolism in animals was limited, autoradiography indicated the presence of a metabolite with an RF value of 0.12.