Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

(2010)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

EC Number:

266-358-7

EC Name:

Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

Cas Number:

66423-13-0

Molecular formula:

C18H27ClO3

IUPAC Name:

octyl (2R)-2-(4-chloro-2-methylphenoxy)propanoate

Constituent 2

Reference substance name:

Mecoprop-P n-octyl ester

IUPAC Name:

Mecoprop-P n-octyl ester

Constituent 3

Reference substance name:

Preventol B5

IUPAC Name:

Preventol B5

Constituent 4

Reference substance name:

R-(+)-2-(4-chloro-2-methylphenoxy)-propionicacid, octyl ester

IUPAC Name:

R-(+)-2-(4-chloro-2-methylphenoxy)-propionicacid, octyl ester

Details on test material:

- Stability under test conditions: The stability and homogeneity of a trial formulation was assessed over 24 hours. Comparison of the respective chromatograms showed no degradation of the radiolabelled test article over this time.

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Weight at study initiation for animals in the excretion balance and tissue distribution investigations: 231-281 g; Weight at study initiation for animals in the pharmacokinetic investigations: 268-288 g; The body weights of animals within each dose group were within 20% of the mean body weight.
- Fasting period before study: no
- Housing: in groups up to 5/cage if not used for excretion balance study
- Individual metabolism cages: yes; for excretion balance study the animals were placed in individual glass metabolism cages suitable for the separate collection of urine and faeces following the administration of the radiolabelled substance
- Diet and water: ad libitum
- Acclimation period: at minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 32-74
- Air changes (per hr): at minimum 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of radiolabelled and non-radiolabelled test substance were co-dissolved in acetonitrile and the solvent removed under nitrogen convection. The test substance was then suspended in 1% aqueous carboxymethylcellulose. A small volume of Tween 80 (<0.05% of the formulation weight) was added to the formulations as a wetting agent.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The stability and homogeneity of a trial formulation was assessed over 24 hours. Comparison of the respective chromatograms showed no degradation of the radiolabelled test article over this time. The purity of the dosing formulations was assessed by subjecting portions of the formulations to HPLC analysis pre-dose administration. The homogeneity of the formulated test substances was checked by subjecting replicate aliquots of the formulations to LSC pre and post dose administration. The radioactive concentration of the replicates varied by less than 8% and the formulations were considered homogeneous.
The specific activity of the formulated test substance for the pharmacokinetic and for the excretion balance study was 0.863 MBq/mg.
The specific activity of the formulated test substance for the tissue distribution study was 1.028 MBq/mg.

Duration and frequency of treatment / exposure:

single administration

Doses / concentrations

Remarks:

Doses / Concentrations:
5 mg/kg

No. of animals per sex per dose / concentration:

pharmacokinetic study: 4 males
excretion balance study: 4 males
tissue distribution study: 16 males

Control animals:

no

Details on study design:

- Study duration 120 h for pharmacokinetic study and 168 h for excretion balance study

- Study design: Following a single oral administration of [14C]-Mecoprop-P n-octyl ester, blood samples were taken from four male rats at various times up to 120 hours. A portion of blood was used to prepare plasma.
A further group of four male rats were placed in metabolism cages. Urine and faeces were collected at intervals up to 168 hours. Expired air was collected up to 24 hours. At 168 hours, selected tissues were excised or sampled.
An additional group of four male animals were sacrificed at 4, 38, 72 and 96 hours and selected tissues were excised or sampled.
Urine, faeces, plasma, liver and kidneys were subjected to metabolite profiling and selected samples to metabolite identification.

The radioactive concentration in all samples was determined by liquid scintillation counting (LSC).

- Further examinations:
All animals were examined at the beginning and the end of the working day, to ensure the animals are in good health. Body weights were recorded the day after arrival, before dose administration and at necropsy, where applicable.

- Dose selection rationale: The dose was chosen based on a toxicokinetic study of two structurally related substances (Lappin, 1997).

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum and other tissues (Adrenals, Bone, Bone marrow, Brain, Epididymis, Eyes, Fat, GI Tract & contents, Hair and Skin, Heart, Kidneys, Liver, Lungs, Muscle (quadriceps), Pancreas, Pituitary gland, Spleen, Testes, Thymus, Thyroid, Carcass, Blood, Plasma, Red blood cells, and residual carcasses), expired air, cage debris, and cage washes.
- Time and frequency of sampling: blood sampling at 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48, 72, 96 and 120 hours post dose, urine collection pre-dose and at 6, 12, 24, 48, 72, 96, 120, 144 and 168 hours post dose, faeces collection at 24 hour intervals until 168 hours after dosing, and expired air was collected at 24 hours post dose. At each collection of excreta, cage debris was removed and the cages rinsed with a small volume of water. Cage debris was pooled separately for each animal over the 168 hour collection period. Cage washings were collected separately. After the final water wash, the cages were washed with methanol which was added to the final water wash.
- Other: Radioactivity was determined in blood, plasma, urine, faeces, cage debris, cage washings, expired air, tissues and residual carcass.

TISSUE DISTRIBUTION STUDY
- Tissues and body fluids sampled: blood, plasma, and other tissues (Adrenals, Bone, Bone marrow, Brain, Epididymis, Eyes, Fat, GI Tract & contents, Hair and Skin, Heart, Kidneys, Liver, Lungs, Muscle (quadriceps), Pancreas, Pituitary gland, Spleen, Testes, Thymus, Thyroid, Carcass, Blood, Plasma, Red blood cells, and residual carcasses).
- Time and frequency of sampling: At 4, 38, 72 and 96 hours post-dose, four animals each were sacrificed.
- Other: Radioactivity was determined in blood, plasma, carcasses and tissues.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, plasma, tissues (liver and kidneys), cage washes, bile
- Time and frequency of sampling: Samples of urine (6, 12, 24 and 48 hour samples), faeces (24 and 48 hour samples), plasma (4, 38, 72 and 96 hour samples), liver (4, 38, 72 and 96 hour samples) and kidneys (4, 38, 72 and 96 hour samples) were pooled by matrix and time point. Tissues were pooled by taking a percentage of the sample weight and plasma by equal weights.
- Method type(s) for identification: Extracts were examined for evidence of metabolism using HPLC with radiodetection (HPLC column Spherisorb ODS2 (250 x 4.6mm, 5μm particle size). Selected samples were analysed by LC/MS in order to structurally elucidate selected metabolites. Negative ion electrospray mass spectrometric analysis was carried out with a capillary temperature of 290°C, and a spray voltage of 4.5 kV. The scan range was m/z 100 to 800.
- Limits of detection and quantification: The limit of quantification for the analysis of each sample type by liquid scintillation counter was taken as twice the background disintegration rate obtained from the measurement of blank samples.

Statistics:

Liquid scintillation counts and weighing data were either entered manually or captured on-line using a validated data acquisition system (Debra version 5.7.8.124, LabLogic Ltd). HPLC data were captured on line with Laura software (version 3, LabLogic Ltd). This software package was used to integrate the area under the trace and express each region of interest as a fraction of the total number of counts detected. Where appropriate, fractions were collected over 12 seconds and subjected to LSC by a TopCount NXT Microplate Scintillation Counter. The LSC data were imported into Laura for the reconstruction of radiochromatograms. Mass Spectrometry data were captured on-line and evaluated using a validated data acquisition system (Xcalibur v 2.0.7, ThermoFisher).

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Absorption was rapid and extensive (>95% of the dose), with detectable concentrations of radioactivity in the plasma by 0.25 hours post dose and maximum concentrations within 4.5 hours.

Main ADME results 2

Type:

distribution

Results:

Distribution of radioactivity was rapid and extensive by 4 hours after dosing. The majority of tissues contained concentrations greater than those in plasma (systemic exposure). There was no evidence of accumulation of radioactivity in tissues.

Main ADME results 3

Type:

metabolism

Results:

There were two common major metabolites: One is formed by de-alkylation of the test substance resulting in the loss of the octane chain (45% of the dose), the second is formed by hydroxylation of the first metabolite (37% f the dose).

Main ADME results 4

Type:

excretion

Results:

The elimination of radioactivity was rapid; >92% of the administered dose being excreted via urine within 24 hours. Clearance of radioactivity was slow (t½=34.1 h), with concentrations being detected in the majority of sampled tissues at 168 hours.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

- Pharmacokinetic study:
Animals were exposed to Preventol B5 at 0.25 hours post-dose, indicating rapid absorption. Mean maximum plasma concentrations of radioactivity were achieved within 4.5 hours of dose administration.

- Excretion balance study:
Absorption was extensive at >95% of the dose, calculated from excretion in urine and recovery of radioactivity in carcass and tissues.

Details on distribution in tissues:

- Pharmacokinetic study:
The blood:plasma ratio indicated that there was no association of the test substance or its radiolabelled metabolites with the cellular fraction of the blood. Blood concentrations mimicked those in plasma but were approximately one half their value.

- Excretion Balance Investigation:
The majority of tissues contained concentrations greater than those in plasma (systemic exposure). The highest concentrations were in the fat, hair and skin, liver and kidneys. The only tissues with concentrations of radioactivity lower than the plasma were the muscle and the testes. Only the bone, bone marrow, brain, eyes, pituitary, red blood cells and thyroid contained concentrations below the limit of quantification. None of the tissues were considered to contain substantial amounts of radioactivity.

- Tissue Distribution Investigation:
An examination of the concentrations of radioactivity in the tissues and organs of rats showed that the distribution of radioactivity was rapid and extensive by 4 hours after dosing. Radioactivity was rapidly depleted, falling by ca 9 to 434-fold within one half-life following administration of test substance and then falling more slowly, by ca 14 to 323-fold, at the last time point compared with the first sampling time. The greatest concentrations of radioactivity were generally associated with the liver and kidneys, the organs of metabolism and excretion.There was no evidence of accumulation of radioactivity in tissues.

Details on excretion:

- Pharmacokinetic study:
Concentrations of radioactivity were still detectable at 120 hours. The mean t½ was determined as 34.1 hours. The calculated AUCt values were >99% of the extrapolated AUC values indicating almost complete clearance from the plasma.

- Excretion Balance Investigation:
The recovery of radioactivity was essentially quantitative by 168 hours post-dose with the majority (>92%) excreted in the urine. Faecal elimination was ca 4% of the administered radioactivity. Radioactivity recovered from the cage washes and cage debris accounted for <5% of the administered dose. Excised tissues accounted for <0.1% of the administered dose with <0.4% remaining associated with the residual carcass. The greater portion of the radioactive dose (>96%) was recovered within 24 hours of dose administration.
The elimination of radioactivity was rapid, with >92% of the administered dose being excreted within 24 hours. Clearance of radioactivity was slow, with concentrations being detected in the majority of sampled tissues at 168 hours post dose.

Toxicokinetic parameters

Toxicokinetic parameters 1

Toxicokinetic parameters:

Cmax: 20505 ng equivalents/g

Toxicokinetic parameters 2

Toxicokinetic parameters:

Tmax: 4.5 hours

Toxicokinetic parameters 3

Toxicokinetic parameters:

half-life 1st: 34.1 hours

Toxicokinetic parameters 4

Toxicokinetic parameters:

other: AUCt 214481 ng equivalents.h/g

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

- Metabolic Profile:
Metabolism of the substance was quantitatively extensive, but limited in scope. There were two common major metabolites in liver, kidney, urine, faeces and plasma. One of these metabolites, which accounted for 45% of the dose, is formed by de-alkylation of the test substance resulting in the loss of the octane chain. The second metabolite is formed by hydroxylation of the first metabolite (hydroxylation of the 4-methyl group on the phenoxy ring). This metabolite accounted for 37% of the dose.
Radio chromatograms of pooled plasma taken 4 hours after substance administration onwards revealed no detectable amount of the unchanged Mecoprop-P n-octyl ester. Only one main metabolite was seen in plasma after 4, 38, 72, and 96 hours, which is the ester hydrolysis product Mecoprop-P acid (MCPP-P acid). In liver, kidney, urine, faeces two major metabolites can be detected: Mecoprop-P acid (MCPP-P acid) and a hydroxylation product of Mecoprop-P acid (MCPP-P acid). Again, no detectable amount of Mecoprop-P n-octyl ester was found in these tissues and excretion products. As it could be seen from the time course of the radio chromatograms the amount of hydroxylated Mecoprop-P acid (MCPP-P acid) increases in the liver over time, indicating that the primary ester hydrolysis product Mecoprop-P acid (MCPP-P acid) is further metabolized in the liver. This is supported by the observation, that the highest concentrations of radioactivity were found in liver, amongst few other tissues (fat, hair, skin and kidney).

Any other information on results incl. tables

During the course of the study, no signs of overt toxicological effects, which could have been attributed to the administration of the test substance, were observed in the test animals.

Applicant's summary and conclusion

Executive summary:

A study on toxicokinetics according to OECD TG 417 was conducted with Mecoprop-P n-octyl ester on rats, receiving each orally by gavage a nominal dose of 5 mg/kg. Following substance administration 4 rats were used for determination of pharmacokinetic parameters, 4 rats for excretion balance studies (metabolism cages), and 16 rats for investigation of tissue distribution. Additionally urine, faeces, plasma, liver and kidney were subjected to metabolite profiling.

As result it was found that absorption of the test substance was rapid, with detectable concentrations of radioactivity in the plasma by 0.25 hours post dose and maximum concentrations achieved within 4.5 hours. The plasma half-life was ca 34 hours. Absorption was extensive at >95% of the dose, calculated from excretion in urine and recovery of radioactivity in carcass and tissues. Radioactivity was distributed into the tissues and organs by 4 hours post dose.

Recovery of radioactivity was quantitative by 168 hours post-dose, with excretion mainly via the urine. The elimination of radioactivity was rapid, with >92% of the administered dose being excreted within 24 hours. Clearance of radioactivity was slow, with concentrations being detected in the majority of sampled tissues at 168 hours post dose.

The greatest concentrations of radioactivity were generally associated with the liver and kidneys, the organs of metabolism and excretion. There was no evidence of accumulation of radioactivity in tissues.

Metabolism of Mecoprop-P n-octyl ester was quantitatively extensive, but limited in scope. There were two major metabolites common to urine, faeces, plasma, liver and kidney. One [Mecoprop-P acid (MCPP-P acid)] was formed by de-alkylation and loss of the octyl side chain, the second metabolite was formed by hydroxylation of the first metabolite.