Desmedipham

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-06-10 to 2015-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

This study is new. No OECD test guideline is available for this study. The study is acceptable.

Objective of study:

metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

The comparative metabolism of [Phenoxy-UL-14C] Desmedipham (14C-Desmedipham) was investigated in animal in-vitro systems by incubating the test item with liver microsomes from male Wistar rats (RLM) and humans (HLM) in the presence of NADPH cofactor. The test item concentration was 10 µM and the protein concentration 1 mg/mL. The 10 µM test item concentration was chosen in order to have enough sample material for possible identification of metabolites by chromatographic or spectroscopic methods. The sampling times were 0, 0.5 and 1 hour after test start. The test durations of 0.5 and 1 hour for the test item were considered as reasonable because positive results were obtained from the enzymatic reaction of Testosterone to Hydroxy-Testosterone already after 5 minutes. Samples were analyzed following protein precipitation by reversed phase HPLC with radiochemical detection (HPLC-RAD).

GLP compliance:

yes (incl. QA statement)

Species:

other: Rats and Humans

Strain:

other: Wister Rats and Humans

Sex:

male

Details on test animals or test system and environmental conditions:

Pooled liver microsomes from male Wistar rats (pool of 200 individuals) and humans (pool of 50 donors from both genders)

Route of administration:

application in vitro

Vehicle:

other: Acetonitrile (AcN)

Details on exposure:

IN VITRO APPLICATION

- Concentration of test material and reference chemical: Final concentrations of the incubates were: 5 mM MgCl2; 1 mg/mL microsome protein; 10 µM 14C-Desmedipham (0.133 µCi/incubate); 1 mM reduced NADPH.

The final concentrations were 5 mM MgCl2, 0.15 mg/mL microsome protein, 150 µM testosterone and 1 mM reduced NADPH.

- Method of preparation of stock solution(s) of test material and reference chemical:
Working solution of 14C-Desmedipham (499.5 µM; 13.30 µCi/mL): 30 µL of 14C-Desmedipham stock solution were diluted with 1098 µL of AcN in a 10-mL polypropylene tube and the solution was stirred gently. This solution was freshly prepared and kept at room temperature until use.

1 mM non-Radiolabelled Desmedipham Solution was prepared by weighing 8.28 mg of non-radiolabelled Desmedipham in a 25-mL volumetric flask. The level mark was filled up with AcN, and the solution was stirred until complete dissolution of the solid. This solution was freshly prepared and kept at room temperature until use.

- Cell culture medium characteristics (temperature, pH): Room temperature until the end of the
experiment and were further stored at -80°C±10°C until analysis, pH 7.4

- Incubation temperature: 37 ± 1°C.

- Number of replicates: Triplicate samples at T=0 (R-0-1, R-0-2, R-0-3, H-0-1, H-0-2 and H-0-3; not incubated) were prepared by adding the same components as test samples but in different order (i.e., AcN was added prior to NADPH and 14C-Desmedipham).

- Time points: These samples were incubated for 0.5 and 1h, respectively.

Duration and frequency of treatment / exposure:

These samples were incubated for 0.5 and 1h, respectively.

Dose / conc.:

0 ppm

Remarks:

Buffer control

Dose / conc.:

10 ppm (nominal)

Positive control reference chemical:

Metabolite Standard Used in Positive Metabolism Controls of the In-vitro System:
6ß-hydroxytestosterone.
Internal Standard Use in Positive Metabolism controls of the In-vitro Systems:
Dexamethasone Vetranal

Details on study design:

- Dose selection rationale: The 10 µM test item concentration was chosen in order to have enough sample material for possible identification of metabolites by chromatographic or spectroscopic methods.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- 14C-Desmedipham was incubated separately with RLM and HLM (n=3) at 37±1°C in a final volume of 500 µL. Incubations were performed in a thermomixer device (Eppendorf) with shaking at 1000 rpm. The microsomal incubates were centrifuged at 16000 x g for 15 minutes at 20°C. After centrifugation, 100 µL of each supernatant were diluted with 400 µL of HPLC mobile phase A. The samples were directly analysed by HPLC-RAD without any further extraction procedure.

ANALYTICAL METHOD
- Samples were analysed by HPLC-RAD for the unchanged test item and metabolites using the analytical method listed in the table that is attached in "Overall remarks, attachments".
The chromatograms were recorded electronically and quantitatively evaluated using the MassLynx® Chromatography software (V4.0). The 14C-trace of a chromato-gram, which should be integrated, was divided into regions of interest, corresponding to the separated radioactive peaks. The area counts from all regions of interest were used for the percentage calculation of the individual compounds.

Expression of the Results:
Relative Percentages of Unchanged 14C-Desmedipham and Metabolites: The relative percentages were calculated from the radio chromatographic profiles at the different incubation times.

Testosterone 6ß-hydroxylase Activity in Metabolism Positive Controls: The results from the positive metabolism control incubations will be expressed as testosterone 6ß-hydroxylase enzyme activity (CYP3A).

Type:

metabolism

Results:

The study did not identify any unique human metabolites

Metabolites identified:

yes

Details on metabolites:

Metabolite Profile of 14C-Desmedipham:

14C-Desmedipham showed high instability in the incubation buffer at pH 7.4, in the samples incubated for 0.5 and 1 hours at 37°C. A 14C-labelled product (namely Dm-5) was found after 0.5- and 1-hour incubation. The radioactive peak corresponding to Dm-5 accounted for 53.6% and 76.6% of the total peak areas in the chromatogram. Dm-5 degradation product was also detected in RLM and HLM incubated for 0 hours and accounted for 6.3% and 6.0% of the relative percentage of peak area, respectively, indicating that 14C-Desmedipham was instable under the microsomal test conditions. 31.1% and 13.4% of 14C-Desmedipham remained unchanged when incubated with RLM for 0.5 h and 1 h, respectively. In these incubations, a total of eight 14C-labelled potential metabolites could be detected in addition to compound Dm-5 (namely Dm-1, Dm-2, Dm-3, Dm-4, Dm-6, Dm-7, Dm-8 and Dm-9).

At both incubation time periods, three of the above mentioned 14C-labeled metabolites were detected below the LLOQ and three of them accounted for mean 5% or more of the relative percentage at 0.5 h incubation: Dm-1 (6.8%), Dm-6 (5.4%) and Dm-7 (16.8%) and only two at 1 h incubation: Dm-1 (13.4%) and Dm-7 (14.7%). The remaining metabolites (Dm-3, Dm-8 and Dm-9) showed mean relative percentages ranging from 0% to 2.8%. The relative percentages of the degradation product Dm-5 were 38.8% and 55.7% after 0.5- and 1-hour incubations, respectively, in RLM. These percentages were remarkably lower as compared with the ones in the samples incubated in buffer only, meaning that Dm-5 is subsequently metabolized in the RLM system. Figure 2 shows the representative HPLC-RAD profiles of 14C-Desmedipham in RLM.

44.0% and 23.5% of 14C-Desmedipham remained unchanged when incubated with HLM for 0.5 and 1 hours, respectively.

In these incubations, a total of 6 14C-labelled potential metabolites could be detected in addition to compound Dm-5 (namely Dm-1, Dm-3, Dm-4, Dm-6, Dm-7, and Dm-9). The rat metabolites Dm-2 and Dm-8 were not found at detectable levels in HLM. Two of the above mentioned 14C-labeled metabolites accounted for mean 5% or more of the relative percentage: Dm-6 (6.2% at 0.5 h and 8.0% at 1 h) and Dm-7 (11.6% at 0.5 h and 12.2% at 1 h). The remaining metabolites showed mean relative percentages ranging from 0% to 4.5%. The relative percentages of the degradation product Dm-5 were 37.6% and 49.8% after 0.5- and 1-hour incubation, respectively, in HLM. These percentages were lower as compared with the ones in the samples incubated in buffer only, meaning that Dm-5 is also subsequently metabolized in the HLM system. No human-specific metabolites of 14C-Desmedipham were found in the liver microsomes system in the present study.

The results of the present in vitro study demonstrated a slightly different metabolic pattern of 14C-Desmedipham when comparing rat and human liver microsomes that were incubated with the test item for 0.5 h and 1 h. This observation belongs however only to the lower number of metabolites detected in HLM. All metabolites, of which the two largest (Dm-6 and Dm-7) must be specifically mentioned, were also detected in the tests with rat liver microsomes.

Peak areas were recorded for each chromatogram. The LLOQ value was set at the 500 dpm level for radioactivity detection (cv <20%). These results indicate that after analysis of test samples, compounds showing radioactive peak areas below the mean peak area value obtained for the LLOQ (peak area of 1683.7) were not considered for quantification.

 

 

 

Positive Metabolism Controls

 

Formation of 6β-hydroxytestosterone from testosterone demonstrated sufficient metabolic capability of the microsome batches used in the study. However, due to the short incubation time (5 minutes) it was affected by the lack of the usual 2-minutes preincubation at 37°C mostly in the RLM incubations. Testosterone 6 β-hydroxylase activities was found to be 749.6 pmol/mg/minute in male rat liver microsomes and, 2415.5 pmol/mg/minute in pooled human liver.

 

 

 

Recovery of Radioactivity

 

The mean recovery of radioactivity after microsome incubations and sample preparation (i.e., protein precipitation with AcN and centrifugation) at T=0 hours was found to be 102.1% and 94.4% in RLM and HLM, respectively, after 0.5-hour incubation the recoveries were 97.6% in RLM and 98.2% in HLM, after 1 hour incubation the recoveries were 99.3% in RLM and 96.9% in HLM.

Conclusions:

14C-Desmedipham was highly instable in incubation buffer at 37°C and at pH 7.4. A single degradation product (Dm-5) was formed in incubations with buffer alone accounting for 53.6% of the radioactivity after 0.5 h incubation and 76.6% after 1 h incubation.

In rat and liver microsomes, Dm-5 amounted to 37.6-38.8% after 0.5 h incubation and 49.8-55.7% after 1 h incubation. These percentages were lower as compared with the ones in the samples incubated in buffer only, meaning that Dm-5 is also subsequently metabolized in the rat liver and human liver microsome systems.

The in-vitro metabolite profile of 14C-Desmedipham when incubated with liver microsomes was slightly different between rats and humans.

In incubations with rat liver microsomes, 31.1% and 13.4% of the initial 14C-Desmedipham remained unchanged at 0.5 h and 1 h incubation, respectively. 14C-Desmedipham was metabolized towards a high number of metabolites. Under the experimental conditions used in the present study, a total of 8 metabolites were detected in addition to the degradation product Dm-5, three (Dm-1, Dm-6 and Dm-7) of them were above 5% of the relative percentage at 0.5 h incubation and two (Dm-1 and Dm-7) at 1 h incubation.

In human liver microsomes, 14C-Desmedipham was metabolized to lower number of metabolites (six in addition to compound Dm-5). The percentage of 14C-Desmedipham remaining after 0.5 h and 1 h incubation was slightly higher (44.0% and 23.5%, respectively), indicating a slightly slower metabolism rate of 14C-Desmedipham in human liver microsomes.

From the six detectable metabolites formed by human liver microsomes two (Dm-6 and Dm-7) were the most important compounds because of their higher relative percentage values (from 6.2% to 12.2%). These metabolites were also detected as major metabolites in incubations with rat liver microsomes.

In summary, it can be assumed that in incubations with human liver microsomes not specifically different 14C-Desmedipham metabolites are formed in comparison with rat liver microsomes.

Executive summary:

The comparative metabolism of [Phenoxy-UL-¹⁴C] Desmedipham (¹⁴C-Desmedipham) was investigated in vitro by incubating the test item with liver microsomes from male Wistar rats (RLM) and humans (HLM) in the presence of NADPH cofactor. The test item concentration was 10 μM and the protein concentration 1 mg/mL. The 10 μM concentration was chosen in order to have enough sample material for possible identification of metabolites by chromatographic or spectroscopic methods. The sampling times were 0, 0.5 and 1 hours after test start. The test durations of 0.5 and 1 hours were considered as reasonable because positive results were obtained from the enzymatic reaction of Testosterone to Hydroxy-Testosterone already after 5 minutes. Samples were analyzed following protein precipitation by reversed phase HPLC with radiochemical detection (HPLC-RAD).  The recovery of radioactivity was measured in both microsome incubations and amounted to ≥96.9% for the 0.5- and 1-hour samples.  The metabolic activity of the microsomes was clearly demonstrated by determining 6β-hydroxytestosterone that was formed from testosterone by testosterone 6β-hydroxylase. This biochemical reaction is a well-known marker for the CYP3A microsomal enzyme.  ¹⁴C-Desmedipham was highly instable after incubation with buffer at 37^°C and pH 7.4. A single radiolabeled compound (Dm-5) was produced and accounted for >53.6% of the radioactivity. Dm-5 was also detected in the liver microsome incubations from both species but in lower amounts, meaning that Dm-5 was subsequently metabolized by the liver microsome preparations.  The results of the tests indicated that the in vitro metabolism of ¹⁴C-Desmedipham when incubated with liver microsomes was slightly different between rats and humans.  In rat liver microsomes, 31.1% and 13.4% of the initial ¹⁴C-Desmedipham remained unchanged after 0.5 h and 1 hour incubation, respectively. ¹⁴C-Desmedipham was metabolized towards a high number of metabolites. A total of 8 metabolites were detected in addition to the degradation product Dm-5, three of them were above 5% of the relative percentage.  In human liver microsomes, a total of 6 metabolites were found in addition to Dm-5. The percentage of ¹⁴C-Desmedipham remaining after 0.5 h and 1 h incubation was slightly higher as compared to the rat liver microsomes system indicating a marginally slower metabolism rate of ¹⁴C-Desmedipham in human liver microsomes. From the five detectable metabolites formed by human liver microsomes, two (Dm-6 and Dm-7) were the most important compounds because of their high relative percentage values (from 6.2% to 12.2%). Metabolites Dm-6 and Dm-7 were also detected as major metabolites in incubations with rat liver microsomes.  The slightly different metabolic pattern of ¹⁴C-Desmedipham when comparing rat and human liver microsomes belongs however only to the lower number of metabolites detected in human liver microsomes. All metabolites, of which the two largest (Dm-6 and Dm-7) must be specifically mentioned, were also detected in the tests with rat liver microsomes.  In summary, it can be assumed that in incubations with human liver microsomes not specifically different ¹⁴C-Desmedipham metabolites are formed in comparison with rat liver microsomes. No human-specific metabolites of ¹⁴C-Desmedipham were found in the liver microsomes system.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-03-24 to 1995-03-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Objective of study:

excretion

metabolism

tissue distribution

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

After the study was performed, a new version of the OECD Test Guideline 417 has been adopted 22nd July, 2010. The main study fulfils these data requirements except that AUC and plasma half-life are new data requirements which were not calculated.

Qualifier:

according to guideline

Guideline:

other: EC Guideline OJL133

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

2 Radiolabelled and 1 non-radiolabelled test materials were used.

Radiolabelling:

yes

Remarks:

2 radiolabelled test materials and 1 non-radiolabelled

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat has been chosen as the species for study where this is a species which has been used in the toxicological evaluation of the test material.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9-10 weeks
- Housing: Throughout the acclimatisation period the animals were housed 3 to a cage in polypropylene and stainless-steel cages with wood shavings as bedding. Animals used for the blood kinetics studies were housed individually in polypropylene and stainless-steel cages with raised wire mesh floors to inhibit coprophagy. Animals used for the excretion studies were housed individually in all-glass metabolism cages specifically designed for the separate, quantitative collection of urine and faeces.
- Diet: Standard laboratory diet (SDS Rat and Mouse Maintenance Diet No. 1),ad libitum.
- Water: domestic mains water was available ad libitum
- Acclimation period: 16 days
- Health status: Animals were in good health and suitable for inclusion in the study. No animal was excluded from the study on the basis of ill-health.

ENVIRONMENTAL CONDITIONS
Air temperature and relative humidity were recorded daily throughout the acclimatisation and study periods.

IN-LIFE DATES: From: To: 28 April 1994 to 30 November 1994

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in corn oil, it was expected that the test substance would be soluble in corn oil (and this vehicle has been used in toxicity studies). Two dose levels were used. These dose levels will correspond to a low oral dose (1 mg/kg bw) at which no toxicity is expected and a high oral dose (100 mg/kg bw) which may elicit a mild toxic effect. Each animal received a single oral dose by gavage.

Duration and frequency of treatment / exposure:

Dose was applied once

Dose / conc.:

1 mg/kg bw (total dose)

Remarks:

Single oral low dose

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

Single oral high dose

No. of animals per sex per dose / concentration:

4/sex/group

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
Phase 1: Excretion Kinetics and Tissue Retention (Low Dose Level):
At 168 h post dose animals were humanely sacrificed and the following tissues and organs
retained: Whole blood, Plasma, Heart, Lung, Spleen, Kidneys, Liver, Perirenal fat, Testes/ovaries, Gastrointestinal tract plus contents, Uterus, Muscle (leg), Brain, Thyroid, Adrenals, Skin, Remaining carcass, Bone mineral, Bone marrow.
Total radioactivity was measured in all samples of urine, faeces, cage wash, tissues, and organs.

Phase 2: Excretion Kinetics and Tissue Retention (High Dose Level):
Samples retained for radioactivity analysis as described previously for Phase 1.

Phase 3: Blood Kinetics (Low Dose Level):
Blood samples (target volume ca 0.2 ml) were collected from the tail vein into heparinised tubes at the following target times after dose administration: 0.25, 0.5, 1, 2, 4, 6, 8, 24 and 48 h. The actual times of blood sampling were recorded. Total radioactivity was measured in all samples of whole blood.

Phase 4: Blood Kinetics (High Dose Level):
Samples retained for total radioactivity analysis as described previously for Phase 3.

ANALYTICAL METHOD

Liquid Scintillation Analysis: All samples were analysed for 5 min using a scintillation analyser with automatic quench correction using an external standard method. Representative blank samples were analysed and their values subtracted from the biological sample measurements. The activity in each sample was expressed as net d.p.m. A limit of reliable determination of 30 d.p.m. above background has been instituted in these laboratories. If results arise from data 10-30 d.p.m. above background the fact has been noted in the results section of this report. Similarly, the fact has been noted if results arise from data less than 10 d.p.m. above background.

Combustion Analysis: Samples for combustion were weighed into combust cones and combusted using a Packard Tri-Carb 306 Automatic Sample Oxidiser. The resultant 14CO2 was absorbed in Carbo-Sorb® and mixed automatically with Permafluor®E+ scintillation fluid. Combustion efficiency was checked routinely several times throughout each production run.

Type:

absorption

Results:

At the low dose, based on urinary excretion alone, approximately 80% of the administered dose was absorbed.
At the high dose, based on urinary excretion alone, approximately 65% of the administered dose was absorbed.

Type:

distribution

Results:

Low dose: Mean tissue residues at 168 h post dose accounted for 0.51% in male and 0.72% in female rats.
High dose: Mean tissue residues at 168 h post dose accounted for 1.2% in male and 1.67% in female rats.

Type:

excretion

Results:

In both doses: Mean whole blood concentrations of total radioactivity were higher in female rats. The mean whole blood concentration - time curve in both male and female rats however, followed a similar pattern.

Details on absorption:

Phase 3: Blood Kinetics (Low Dose Level)
Absorption was rapid with the highest mean concentration of total radioactivity in whole blood at 2 h post dose, representing 0.290 µg equiv/g (range 0.220-0.349 µg equiv/g) and 0.503 µg equiv/g (range 0.428-0.581 µg equiv/g) in male and female rats, respectively.

Phase 4: Blood Kinetics (High Dose Level)
The rate of absorption was slower at the high dose level with the highest mean concentration of total radioactivity in whole blood at 8 h post dose representing 37.8 µg equiv/g (range 31.8-42.6 µg equiv/g) and 43.1 µg equiv/g (range 38.9-46.1 µg equiv/g) in male and female rats, respectively.

Details on distribution in tissues:

Following the low dose: Mean tissue residues at 168 h post dose accounted for 0.51% and 0.72% of the administered dose in male and female rats, respectively, the majority of which was associated with the residual carcass. Highest mean concentration of total radioactivity at 168 h post dose were found in whole blood (males = 0.0290 µg equiv/g, females = 0.0485 µg equiv/g) (Table 4). The tissue distribution of total radioactivity at 168 h post dose was independent of gender.

Following the high dose: Mean tissue residues at 168 h post dose accounted for 1.20% and 1.67% of the administered dose in male and female rats, respectively, the majority of which was associated with the residual carcass. Highest mean concentrations of total radioactivity at at 168 h post dose were found in whole blood (males = 8.19 µg equiv/g corresponding to 0.01% of dose, females = 8.77 µg equiv/g corresponding to 0.03 % of dose) (see Table 8). The tissue distribution of total radioactivity was independent of gender.

Details on excretion:

In the low dose group, following administration of [14C]-Desmedipham, urinary excretion was the major route of elimination with a mean of 80.47% (range 73.80-85.60%) and 76.16% of the administered dose (69.25-79.80%) recovered over 0-168 h post dose, in male and female rats, respectively. Based on urinary excretion alone, approximately 80% of the administered dose was absorbed. Over the same period, a mean of 15.09% (11.59-18.10%) and 12.9% of the administered dose (10.00-17.04%) was recovered in faeces in male and female rats, respectively. The amount found in cage wash after 168 hours were 2.28% for males and 3.01% for females.

Excretion was rapid, with a mean of 93.59% and 88.40% of the dose excreted in male and female rats, respectively during the first 24 hours post dose. The routes and rates of excretion were essentially independent of gender. The mean total excreted during 168 hours post dose in male and female rats was 97.84% and 92.11% of the administered dose, respectively with the mean total recovered over the same period 98.35% (range 95.86-100.60%) and 92.84% of the administered dose (range 90.65-95.90%) in male and female rats, respectively.

In the high dose group: Following administration of the test substance, urinary excretion was the major route of elimination with a mean of 71.49% (range 67.67-76.42%) and 63.13% of the administered dose (range 53.59-72.90%) recovered over 0-168 hours post dose, in male and female animals, respectively. Based on urinary excretion alone, approximately 65% of the administered dose was absorbed. Over the same period, a mean of 21.76% (range 16.92-26.29%) and 27.21% of the administered dose (range 17.89-35.83%) was recovered in faeces in male and female rats, respectively. 2.81% and 4.51% of the administered dose were found in cage wash in males and females, respectively.

Excretion was rapid, with a mean of 86.16% and 81.36% of the dose excreted in male and female rats, respectively during the first 24 h post dose. The routes and rates of excretion were essentially independent of gender. The mean total excreted during 168 hours post dose in male and female rats was 96.06% and 94.85% of the administered dose, respectively, with the mean total recovered over the same period 97.27% (range 95.28-98.50%) and 96.52% of the administered dose (range 96.04-97.33%) in male and female rats, respectively.

Test no.:

#1

Toxicokinetic parameters:

Cmax: Cmax (blood) :0.290 µg equiv/g and 0.503 µg equiv/g in male and female rats, respectively. (low dose)

Test no.:

#1

Toxicokinetic parameters:

Tmax: 2 hours (low dose)

Test no.:

#2

Toxicokinetic parameters:

Cmax: (blood): 37.8 µg equiv.g and 43.1 µg equiv/g in male and female rats, respectively. (High dose)

Test no.:

#2

Toxicokinetic parameters:

Tmax: Tmax (blood) : 8 hours (high dose)

Metabolites identified:

yes

Details on metabolites:

Metabolite profiling:
Profiling of urinary metabolites:
In the initial TLC analysis of the 0-6 h urine samples in Solvent System 3 all urinary metabolites remained at the origin, whereas the available metabolite standards all moved. Solvent System 4 was developed to separate the urinary metabolites of desmedipham. Analysis of the pooled urine samples in Solvent System 4 indicated the presence of 3 major metabolites of desmedipham. A similar metabolite profile was observed for both male and female rats, and for both high and low dose levels.

Profiling of faecal metabolites:
Acetonitrile extracted 84-92% of the radioactivity present in the aqueous homogenate of faeces. The proportions of desmedipham metabolites in the faeces extract were determined by TLC in Solvent System 7. Unchanged desmedipham accounted for the majority of the radiolabel present in faeces. HPLC analysis of the extract of the high dose faeces pool confirmed that desmedipham was the major radioactive component in faeces. A minor more polar component was also separated, however it was not possible to separate the minor radioactive components from endogenous material.

Metabolite identification:
Liquid Chromatography Mass Spectrometry (LC-MS) of Urine Samples: The mass spectrometer was tuned in the positive ion electrospray (ESI+) mode using a reference standard of EHPC. The spectrum for EHPC shows prominent ions at m/z 182, 199 and 204 which would correspond to MH+, MNH4+, MNa+ adducts, respectively. Thus, it was predicted the corresponding adduct ions would be detected for the expected desmedipham metabolites.

Quantification of Metabolites:
The proportions of desmedipham metabolites in the urine were determined by HPLC. Three major metabolites; 4-hydroxyacetanilidesulphate (37-46% of urinary activity), EHPC glucuronide (11-22% of urinary activity), and EHPC sulphate (16-21% of urinary activity) were detected. No unchanged desmedipham was detected in urine, however a small proportion (1-4%) of a hydroxylated metabolite, hydroxydesmedipham was detected. 3-hydroxyacetanilidesulphate (<2% of urinary activity) was a minor metabolite. The hydroxyacetanilide isomers were also detected as the corresponding glucuronide conjugates, these were not quantified.

See the Results' tables in the file attached in "Overall remarks, attachments"

Conclusions:

A single oral dose of [14C]-Desmedipham was moderately well absorbed in the rat, and urinary excretion was the major route of elimination. The absorbed dose was extensively metabolised, primarily by hydrolysis. The routes and rates of metabolism were similar in both male and female rats and at both dose levels. Absorption was slightly reduced and delayed at the high dose level. Excretion was rapid and almost complete, and tissue concentrations of radioactivity at 168 h were low. The highest concentration of total radioactivity was in whole blood. The concentration of radioactivity in whole blood at the high dose was 2-4 times higher than would be predicted from the proportional increase in dose.

Executive summary:

In this study an approximately equimolar mixture of separate ring labelled forms of 14C-Desmedipham was administered as a single oral dose to four groups of rats. Each group contained four male and four female rats with two groups receiving doses at the low dose level (1 mg/kg bw) and two groups at the high dose level (100 mg/kg bw). Excreta were collected for 7 days, and tissues retained at 168 hours post dose from one group and serial blood samples collected up to 48 hours post-dose from the second group at each dose level. Samples collected were analysed for total radioactivity and the nature of radioactivity in key urine and faeces samples investigated.  At the low dose, urinary excretion was the major route of elimination accounting for a mean of 80.47% and 76.16% of the administered dose over 168 hours in male and female rats, respectively. Over the same period, a mean of 15.09% and 12.94% of the administered dose was recovered in faeces, in male and female rats, respectively. Excretion was rapid with the routes and rates of excretion essentially independent of gender. The mean total excreted during 168 hours post dose in male and female rats was 97.84% and 92.11% of the administered dose, respectively. Correspondingly tissue residues at 168 hours post dose were low, accounting for less than 1% of the dose. Highest mean concentration of total radioactivity at this time were found in whole blood (males = 0.0290 μg equiv/g, females = 0.0485 μg equiv/g). The tissue distribution of total radioactivity was independent of gender.

 

At the high dose level, urinary excretion was also the major route of elimination accounting for a mean of 71.49% and 63.13% of the administered dose over 168 hours in male and female rats, respectively. Over the same period, a mean of 21.76% and 27.21% of the administered dose was recovered in faeces, in male and female rats, respectively. Excretion was rapid, with the routes and rates of excretion essentially independent of gender. The mean total excreted during 168 hours post dose in male and female rats was 96.06% and 94.85% of the administered dose, respectively.

 

Tissue residues at 168 hours post dose were low, accounting for less than 2% of the administered dose. Highest mean concentrations of total radioactivity at this time were found in whole blood (males = 8.19 μg equiv/g, females = 8.77 μg equiv/g). The tissue distribution of total radioactivity was independent of gender.

 

At both dose levels investigated, mean whole blood concentrations of total radioactivity were higher in female rats. The mean maximum whole blood concentration of total radioactivity following administration at the low dose level was observed at 2 hours post dose, representing 0.290 μg equiv/g and 0.503 μg equiv/g in male and female rats, respectively. Thereafter mean concentrations declined representing 0.054 μg equiv/g and 0.107 μg equiv/g in male and female rats, respectively at 48 hours post dose.

 

At the high dose level, absorption was slower with the mean maximum concentration of total radioactivity in whole blood observed at 8 hours post dose, representing 37.8 μg equiv/g and 43.1 μg equiv/g in male and female rats, respectively.  Thereafter mean concentrations declined representing 10.1 μg equiv/g and 14.4 μg equiv/g in male and female rats, respectively at 48 hours post dose.

 

Three major metabolites were detected in urine following direct LC-MS analysis (representing 70-84% urinary activity), and the major pathway was shown to involve carbamate hydrolysis which yielded different radiolabelled metabolites for the two radiolabel forms.

 

The aniline moiety was mainly excreted as the sulphate of 4-hydroxyacetanilide (37-46% of urinary activity). The hydroxyphenylcarbamate moiety was mainly excreted as EHPC glucuronide (11-22% of urinary activity) and EHPC sulphate (16-22% of urinary activity). Hydroxydesmedipham was detected as a minor urinary metabolite. Other minor metabolites detected were 3-hydroxyacetanilide sulphate arising from the carbamate hydrolysis of EHPC. Glucuronide conjugates of two hydroxyacetanilide isomers were also detected. The metabolite profile in male and female rats was very similar, however comparison of metabolite profiles at the two dose levels revealed a slight change in the relative proportions of metabolites.

 

In conclusion, following a single oral dose of 14C-Desmedipham to rats, urinary excretion was the major route of elimination with the routes of excretion independent of gender. Absorption was essentially independent of gender but was slightly reduced and delayed following administration at the high dose level. Overall, however, excretion was rapid and almost complete. Correspondingly tissue residue of total radioactivity at 168 hours post dose were low with highest concentrations in whole blood. The distribution of total radioactivity in tissues at this time was independent of gender and dose.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-05-12 to 1993-07-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

After the study was performed, a new version of the OECD Test Guideline 417 has been adopted 22nd July, 2010. AUC, Cmax, Tmax were not calculated in this study and exhaled air was not investigated. Distribution in tissues was not investigated.

Objective of study:

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

Two radiolabelled test materials and one non-radiolabelled were not used.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD{SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 148-274 g
- Housing: All animals were housed in Safety Evaluation animal rooms 87 and 93 in PSL 1. After dosing with the radiolabelled test material, the rats were transferred to individual all glass metabolism cages which enabled the separate collection of urine and faeces.
- Diet: pelleted laboratory rodent diet, ad libitum. except for a period of approximately 16 hours prior to dosing with the radiolabelled test material when food was withheld.
- Water: ad libitum.
- Health status: All animals appeared in good health at the commencement of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Photoperiod: 12-hour photoperiod of fluorescent lighting.

Route of administration:

oral: gavage

Vehicle:

other: 1% gum tragacanth in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
PC and EPC radiolabelled forms of desmedipham were prepared at the appropriate specific activities by co-precipitation of radiolabelled and unlabelled desmedipham in methanol. All doses were prepared by suspension of either unlabelled or the radiolabelled desmedipham in an aqueous solution of 1% gum tragacanth.

Exposure: Single low and high dose, single oral dose preceded by 14 consecutive single daily doses of unlabelled desmedipham, orally by gavage.

Duration and frequency of treatment / exposure:

Single low and high dose, single oral dose preceded by 14 consecutive single daily doses of unlabelled desmedipham, orally by gavage.

Dose / conc.:

5 mg/kg bw (total dose)

Remarks:

Single oral low dose: EPC-radiolabel

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Single oral high dose: PC-radiolabel

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Single oral high dose: EPC-radiolabel

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Multiple oral low dose: 14 days with unlabelled followed by a single dose of PC-radiolabel

Dose / conc.:

5 mg/kg bw/day

Remarks:

Multiple oral low dose: 14 days with unlabelled followed by a single dose of EPC-radiolabel

No. of animals per sex per dose / concentration:

5/sex/dose

Control animals:

no

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, cage washes.

- Time and frequency of sampling:
1- EPC and PC radiolabels: single oral high dose studies:
Urine and faeces were separately collected at 24-hour intervals after dosing, for up to 96 hours. After sample collection at 24 hours and 96 hours, the metabolism cages were rinsed down with water. These cage washings were kept separately.

2- EPC radiolabel: single oral low dose studies:
Urine and faeces were separately collected at 0-6, 6-24 and 24-30 hours after dosing. After sample collection at 6 hours and at 30 hours, the metabolism cages were rinsed down with water. These cage washings were kept separately. For the analysis of metabolites in urine the 0-6 hr and 6-24 hr urines and the 6 hr cage washings were pooled for each individual animal.

3- EPC and PC radiolabels: repeated oral dose studies:
Urine, faeces and cage washings were separately collected as described in 2 for the EPC radiolabel and Section 1 for the PC radiolabel. For the EPC radiolabel the urine and cage wash samples were pooled as in 2.

4- All samples were stored at -20°C for the duration of the study, except when sample manipulation was taking place.

- From how many animals: samples pooled, all test animals.

- Method type(s) for identification (GC, HPLC-UV, Liquid scintillation counting, TLC)

- Enzymatic hydrolysis of conjugates using Helix pomatia Juice:
1- Urine samples
A 0.5 ml aliquot of urine was added to 3 ml acetate buffer (0.2 M, pH 5.0) and 0.5 ml Helix pomatia juice (HPJ): Samples were then incubated for at least 16 hr at 37°C in a shaking water bath. Aliquots of blank buffer, buffer and HPJ, and sample incubates were tested for (ß- glucuronidase and aryl sulfatase activity as follows:
i) 2 x 50 µl aliquots were taken from each vial.
ii) To each was added 200 µ1 buffer (Na acetate, 0.2 M, pH 5.0) and either 50 /u.1 phenolphthalein glucuronide (10 mg/ml) or 50 µl catechol sulfate (10 mg/ml).
iii) All samples were incubated for 1 hour at 37°C.
iv) 1-2 drops of 1 M NaOH were then added to each vial and the colour change noted. A bright magenta was indicative of an active (ß- glucuronidase, whilst an active aryl sulfatase was indicated by a deep orange colour.

Methanolic extracts could be deconjugated in a similar manner by evaporating off the methanol from the sample first, then adding the buffer and HPJ. The remainder of the procedure as above.

- Use of DEBRA database:
Weights (pots, tissues, homogenates, aliquots) were either captured directly from balances by a DEBRA 4 database system, recorded manually onto raw data sheets and then edited onto the system or manually entered via a keyboard directly onto the DEBRA 4 database running on a Novell network consisting of Compaq 286/386 computers. Scintillation counts were stored on magnetic disk and subsequently transferred to the system. Printouts from balances and scintillation counters were retained as raw data.
All balance study results, and final data were calculated automatically by the system.

Type:

metabolism

Results:

The major metabolite from the EPC ring radiolabelled desmedipham was N-(3-hydroxyphenyl)ethyl carbamate, which was present in free form in faeces and as conjugates (glucuronic acid and sulfate) in urine.

Metabolites identified:

yes

Details on metabolites:

The major metabolite from the EPC ring radiolabelled desmedipham was N-(3-hydroxyphenyl) ethyl carbamate, which was present in free form in faeces and as conjugates (glucuronic acid and sulfate) in urine. 3-acetamidophenol was the other major metabolite, along with minor amount of 3-aminophenol. These metabolites were present mainly as sulfate and glucuronic acid conjugates. Parent desmedipham was not excreted in urine but was detected in faeces.

A comparison of the high and low dose showed an increased amount of the dose remaining in faeces following the high dose (1000 mg/kg bw). Repeated oral dosing with 5 mg/kg bw EPC ring radiolabelled desmedipham resulted no change in urinary metabolites, but the faeces contained less parent compound (Table 1).

The major urinary metabolite from the PC ring radiolabelled desmedipham was 4-acetamidophenol with minor amounts of 4-aminophenol, 3-aminophenol and 3-acetamidophenol (Table 2). These metabolites were present mainly as glucuronic acid and sulfate conjugates in urine. The major faecal metabolite was N-(phenyl)methyl carbamate, which was not detected in urine. In faeces, parent desmedipham was also present, especially in rats which received the high dose of PC ring radiolabelled desmedipham (1000 mg/kg bw).

Abbreviations:
Desmedipham: DES
N-(3-hydroxyphenyl) ethyl carbamate: Carb
N-(3-hydroxyphenyl) hydroxyethyl carbamate: OH-Carb
N-(phenyl) methyl carbamate: PMC
3-aminophenol: 3-AP
4-aminophenol: 4-AP
3-acetamidophenol: 3-AA
4-acetamidophenol: 4-AAP

See the results' tables attached in "Overall remarks, attachments"

Conclusions:

The major metabolites of the EPC and PC ring radiolabelled desmedipham, N-(3-hydroxyphenyl) ethyl carbamate and 4-acetamidophenol were mainly present in the free form in faeces and as glucuronic acid and sulfate conjugates in urine. A higher amount of the dose was excreted in faeces from the high dose (1000 mg/kg bw) than the low dose (5 mg/kg bw). The amount of the parent compound was highest in faeces at the high dose. There were no apparent sex differences in metabolism in rats which received PC or EPC radiolabelled test material.

An attempt was made to identify the polar material in these samples. In faeces, the results indicated that desmedipham was at least partially broken down and the polar material degraded to compounds of unknown identity. They were possibly formed from aromatic amines, including aniline reacting with each other. However, aniline was not detected in any of the samples analysed. In urine, the polar material probably was at least partly N- glucuronides of 4- and 3-aminophenols.

Executive summary:

SUMMARY AND CONCLUSIONS

1.1 Objective:

The purpose of this study was to determine the metabolic profile of desmedipham in the rat following dosing at a low and high dose and following repeated dosing, using compound radiolabelled in two positions.

 

1.2 Methods:

Urine and faeces from orally dosed rats were appropriately extracted and enzyme hydrolysed. The samples were chromatographed against authentic standards and quantified using HPLC. Some metabolites were identified using mass spectrometry.

 

1.3 Results:

1.3.1 The initial step in the metabolism of desmedipham is the hydrolysis of the carbamate bond between the two rings.

 

1.3.2 The major metabolite from EPC labelled desmedipham is N-(3-hydroxyphenyl) ethyl carbamate which is present in the free form in faeces and as conjugates, with glucuronic acid and sulfate, in the urine.

 

1.3.3 3-Acetamidophenol was the other major metabolite from the EPC labelled desmedipham, along with a minor amount of 3-aminophenol. These were both present mainly as sulfate and glucuronic acid conjugates.

 

1.3.4 The major faecal metabolite from PC labelled desmedipham was phenylmethyl carbamate. This was not found in the urine.

 

1.3.5 The major urinary metabolite from PC labelled desmedipham was 4-acetamidophenol which was present mainly as conjugates with glucuronic acid and sulfate.

 

1.3.6 Minor metabolites from PC labelled desmedipham include 4-aminophenol, 3-aminophenol and 3-acetamidophenol. These were present mainly as glucuronic acid and sulfate conjugates.

 

1.3.7 Parent desmedipham is not excreted in the urine but was seen in the faeces following all of the dosing regimens used.

 

 

1.3.8 A comparison of the high (1000 mg/kg) and low (5 mg/kg) dose studies for the EPC labelled desmedipham showed a higher percentage of the dose excreted in the faeces following the high dose. Urinary metabolites were very similar whilst the low dose study also had minor faecal metabolites not seen in the high dose.

 

1.3.9 A comparison of the high (1000 mg/kg) and low (5 mg/kg) dose studies for the PC labelled desmedipham showed that they had a similar pattern of metabolites, with a higher percentage of the dose being excreted in the faeces after the high dose.

 

1.3.10 Repeated oral dosing with 5 mg/kg prior to an EPC labelled dose resulted in no change to urinary metabolites, but the faeces contained less parent compound with an increased amount of N-(3-hydroxyphenyl) ethyl carbamate.

 

1.3.11 Repeated oral dosing with 5 mg/kg prior to a PC labelled dose resulted in no change to urinary metabolites and faecal metabolites were similar.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-05-12 to 1993-07-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

After the study was performed, a new version of the OECD Test Guideline 417 has been adopted 22nd July, 2010. The main study fulfils these data requirements except that AUC and plasma half-life are new data requirements which were not calculated in this study and exhaled air was not investigated.

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

Two radiolablled test materials and one non-radiolabelled were used in this study

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adults 6-8 weeks
- Weight at study initiation: 150-274 g.
- Housing: After dosing with the radiolabelled test material the rats were transferred to individual all glass metabolism cages which enable the separate collection of urine and faeces.
- Diet: pelleted laboratory rodent diet, ad libitum. except for a period of approximately 16 hours prior to dosing with the radiolabelled test material when food was withheld.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Photoperiod: 12-hour photoperiod of fluorescent lighting.
- Fasting period: 16 hours prior to dosing.

Route of administration:

oral: gavage

Vehicle:

other: 1% gum tragacanth in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
PC and EPC radiolabelled forms of desmedipham were prepared at the appropriate specific activities by co-precipitation of radiolabelled and unlabelled desmedipham in methanol. All doses were prepared by suspension of either unlabelled or the radiolabelled desmedipham in an aqueous solution of 1% gum tragacanth.

Duration and frequency of treatment / exposure:

Single low dose: 5 mg/kg bw 14C-EPC ring.

Single high dose: 1000 mg/kg bw 14C-EPC ring* or 14C-PC ring*

Repeated oral dose: 5 mg/kg bw unlabelled desmedipham (14 days followed by a single labelled dose)

Single oral dose: 5 mg/kg bw 14C-EPC ring* or 14C-PC ring*

* Diluted with unlabelled desmedipham in methanol.

Dose / conc.:

5 mg/kg bw (total dose)

Remarks:

Single oral low dose: EPC-radiolabel

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Single oral high dose: PC-radiolabel

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Single oral high dose: EPC-radiolabel

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Multiple oral low dose (14 days) unlabelled followed by a single low dose: PC-radiolabel

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Multiple oral low dose (14 days) unlabelled followed by a single low dose: EPC-radiolabel

No. of animals per sex per dose / concentration:

5/sex/dose

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC (Absorption, distribution, excretion)
Samples of all excreta, cage washes and tissues were prepared for analysis in triplicate where possible.

- Tissues and body fluids sampled: urine, plasma, cage washes, bone, blood, Adrenals, eyes, ovaries, muscle, fat, Gastro-intestinal tract and carcass, Faeces, and all other tissues.

- Time of sampling: excreta were collected at 6, 24, 48, 30, 72 or 96 hours post radiolabelled dose until over 90% of the dose had been excreted tissue residues 30 or 96 hours after dosing.

- Preparation of samples for quantification of radio activity:
i) Urine (diluted with water when necessary) and plasma: Aliquots (0.1-0.5 ml) were added directly to Scintran FHV scintillant for liquid scintillation counting.

ii) Bone and blood: Weighed samples of bone (approximately 0.1 g) and aliquots of blood (0.25 ml) were combusted in a sample oxidiser and the UCO: trapped in Carbosorb/Permafluor for measurement of radioactivity.

iii) Adrenals, eves, ovaries, muscle, and fat: Whole organs or weighed aliquots were taken and solubilised in SHT (1 ml) at approximately 50°C for up to 48 hours, acidified with hydrochloric acid (5 N, 1 ml) and mixed with Scintran FHV scintillant.

iv) Gastro-intestinal tract and carcass: Digested in sodium hydroxide (10 N) at room temperature for up to 4 weeks. After homogenisation they were sampled, acidified with hydrochloric acid (5 N, 2 ml) and mixed with Scintran FHV scintillant.

v) Faeces, cage wash and all other tissues: The total sample was homogenised in water (some faeces homogenate required further dilution with water) and weighed aliquots (0.25-1.0 ml) were taken, solubilised and prepared for scintillation counting as in (iii) above.

- Quantification of radioactivity:
Radioactivity was measured by liquid scintillation counting with automatic external standard quench correction. Corrected dpm of less than the background cpm were considered to be below the limit of detection.

- Use of DEBRA database:
Weights (pots, tissues, homogenates, aliquots) were either captured directly from balances by a DEBRA 4 database system, recorded manually onto raw data sheets and then edited onto the system or manually entered via a keyboard directly onto the DEBRA 4 database running on a Novell network consisting of Compaq 286/386 computers. Scintillation counts were stored on magnetic disk and subsequently transferred to the system. Printouts from balances and scintillation counters were retained as raw data.
All balance study results, and final data were calculated automatically by the system.

Type:

absorption

Results:

Absorption at the low dose level was 63-83% and was unaffected by repeated exposure. Absorption was lower (33-43%) at the high dose level.

Type:

distribution

Results:

Tissue residue levels were generally low, but highest in the blood and plasma and were markedly higher with the PC label compared to the EPC label, and were slightly higher in females compared to males.

Type:

excretion

Results:

EPC: over 99% was excreted within 30 hours.
Repeated low PC dose: between 86 - 90% was excreted within 24 hours, and over 95 % of the dose within 4 days, mainly in urine (74%).
Single high dose PC: 93% was excreted in 96h, mainly in faeces (56%)

Details on absorption:

EPC-radiolabelled desmedipham was well absorbed after single or repeated low doses (5 mg/kg bw). In rats receiving a single or repeated doses of EPC-radiolabelled desmedipham about 80% of the radioactive dose was excreted in urine within the first 30 hours. In rats, which received a single high dose (1000 mg/kg bw) only about 43% of the radioactive dose was excreted in urine (and cage wash) in the first 96 hours.After repeated low dosing (5 mg/kg bw), between 63 and 77% of PC-radiolabelled desmedipham was excreted in urine within the first 24 hours (the first time point). After the high dose, only about 33% of PC-radiolabelled dose was excreted in urine in the first 96 hours.

Details on distribution in tissues:

Residues were determined at 30 hours (EPC) and 96 hours (EPC, PC) after dosing from the following tissues: adrenals, blood, bone, brain, carcass, eyes, G.I tract, heart, kidney, liver, lungs, muscle, ovaries, plasma, renal fat, spleen and testes. The residue levels were low after single and repeated dosing with EPC ring radiolabelled desmedipham (5 mg/kg bw). The highest mean residue level was found in the gastro-intestinal tract at 30 hours post dose and very low residue levels were found in blood, plasma (0.01-0.02 mg eq/kg tissue), adrenals, liver, testes, and ovaries. The majority of the selected tissues contained detectable levels of radioactivity 96 hours after a single high dose of EPC-radiolabelled desmedipham (1000 mg/kg bw). The highest levels of radioactivity were present in blood (3-4 mg eq/kg tissue) and lesser extent in plasma (0.5-0.7 mg eq/kg tissue) and in liver, heart, kidneys, and carcass. Lower residue levels were found in adrenals, bone, lungs, spleen, renal fat, muscle, gastro-intestinal tract, ovaries and in testes. All selected tissues contained quantifiable levels of radioactivity 4 days after repeated dosing with the PC-radiolabelled desmedipham (5 mg/kg bw). The highest mean radioactivity levels were found in blood and plasma (0.4-1.0 mg eq/kg tissue). Lower residue levels were found in adrenals, bone, brain, eyes, heart, kidney, liver, lungs, spleen, muscle, renal fat, ovaries and in testes. Tissue residue levels in all selected tissues were considerably higher after a single high dose of PC-radiolabelled desmedipham (1000 mg/kg bw). The highest mean levels of radioactivity were found in blood and plasma (122-170 mg eq/kg tissue) 4 days after dosing. The average concentrations of radioactivity were also quite high in lungs, liver, kidney, heart, spleen, muscle, carcass and in ovaries and lower levels of radioactivity were found in adrenals, bone, brain, eyes, renal fat and testes. In general, the tissue residue levels in females were higher than in males. After a single high oral dose of PC ring radiolabelled desmedipham, the terminal blood levels were very high, 123 mg eq/kg in males and 171 mg eq/kg in females, determined 4 days after a high dose (1000 mg/kg bw). Therefore, the apparent lower level of absorption compared to EPC-radiolabelled desmedipham was presumably due to a slower excretion of PC-radiolabelled side of the molecule.

Details on excretion:

In rats which received single or repeated low doses of EPC-radiolabelled desmedipham (5 mg/kg bw), over 99% of the administered dose was excreted within 30 hours. The majority of the radioactivity was eliminated in urine; 80% of the EPC-radiolabelled doses within the first 30 hours. In rats which received a high dose of EPC-radiolabelled desmedipham (1000 mg/kg bw) the excretion was 86% of the dose within 24 hours and nearly 100% within 96 hours. The majority of radioactivity was eliminated in faeces (51%) and the remaining part (43%) in urine. In rats which received repeated low doses of PC-radiolabelled desmedipham (5 mg/kg bw), between 86-90% of the administered dose was excreted within 24 hours, and over 95 % of the dose within 4 days, mainly in urine (74%). After a single high dose (1000 mg/kg bw), 93% of PC-radiolabelled desmedipham was excreted within 96 hours, but the majority of radioactivity was excreted via faeces, 56% and lesser in urine, 33%.

Metabolites identified:

no

See the results' tables attached in "Overall remarks, attachments"

Conclusions:

Desmedipham was absorbed well from the gastrointestinal tract of rats. At single or repeated low dose level (5 mg/kg bw), 77-83% of the EPC-radiolabelled dose was absorbed within the first 6 hours and 63-77% of PC-radiolabelled dose within the first 24 hours. After a high dose (1000 mg/kg bw), the absorption of desmedipham was less complete, only about 43% of EPC- and 33% of PC-radiolabelled dose was excreted in urine in the first 96 hours. The excretion of PC-radiolabelled side of desmedipham molecule was slower than the EPC side of the molecule. After a single or repeated low doses of EPC-radiolabelled desmedipham the residue levels in tissues were very low. After a high dose level (1000 mg/kg bw), residues were detected mainly in blood and plasma (3.7-4.7 mg eq/kg) and in liver, kidneys, lungs, spleen and reproductive organs. Much higher residue levels in these tissues were observed 4 days after PC-radiolabelled desmedipham e.g. in blood and plasma (120-170 mg eq/kg). Thus after a high dose of PC-radiolabelled desmedipham, the residues levels in blood were between 30-36 times and in plasma between 160-180 times higher than after a similar dose of EPC-radiolabelled desmedipham. In general, the mean tissue residue levels were seen to be slightly higher in females than in males.

Executive summary:

The study examined and compared the absorption, distribution, and excretion of radiolabelled residues in the rat following single or repeated oral dosing with either ethyl phenyl (U)-14C carbamate (EPC) or phenyl (U)-14C carbamate (PC) ring radiolabelled desmedipham at dos elevels of 5 and 1000 mg/kg bw.  Male and female rats were dosed orally by gavage with an aqueous suspension of either EPC or PC radiolabelled desmedipham by the following dosing regimens:

Single oral dose - nominally 5 mg/kg/bw (EPC radiolabel only)

Single oral dose - nominally 1000 mg/kg bw

Repeated oral dose of unlabelled desmedipham (nominally 5 mg/kg bw) for 14 consecutive days followed by a single radiolabelled dose

 

The animals were maintained in all glass metabolism cages until over 90% of the dose had been excreted (30 or 96 hours depending on radiolabel and dosing regimen) during which the excretion of the radiolabelled dose in the urine and faeces was monitored.

 

Following dosing with the EPC label, at least 91% of a 5 mg/kg bw dose was absorbed, as evidenced by urinary (plus cage wash) excretion with over 33% (males) and 77% (females) of the dose being excreted in urine (and cage wash) within the first 6 hours, showing that absorption was very rapid. Following the 1000 mg/kg dose, at least 49% (males) and 51% (females) of the dose was absorbed, as evidenced by urinary (plus cage wash) excretion. This also indicates a threshold level for absorption was reached. Following the 1000 mg/kg bw PC-labelled dose, the terminal blood concentrations, were 122.9 mg/kg (males) and 170.9 mg/kg (females), which is further evidence for an extensive absorption of desmedipham occurring.  Tissue distribution levels obtained in both the low dose (5 mg/kg bw) studies (single and repeat dose) with the EPC radiolabel were similar and generally below the LOD 30 hours after dosing. Only gastro-intestinal tract (mean tissue residues in the range 0.03-0.20 mg eq/kg), adrenals, eyes, liver, and carcass (mean tissue residues in the range 0.02-0.06 mg/kg) contained significant residues. Following dosing at the high dose (1000 mg/kg bw) the highest residue levels at 96 hours were present in the blood (3.71 ± 0.65 mg/kg males, 4.74 ± 1.18 mg/kg females) and carcass (3.88 ± 1.12 males, 4.05 ± 1.01 females). In general tissue residue levels in females were greater than in males.  At 96 hours following dosing with the PC-radiolabel in both the low dose (5 mg/kg bw) studies (single and repeat dose), the level and pattern of tissue distribution levels were very similar. The highest tissue residue levels were found present in the blood (mean residue levels in the range 0.49-1.03 mg equivalents desmedipham/kg) and plasma (mean residue levels in the range 0.38-0.76 mg/kg). Following dosing at the high dose level (1000 mg/kg bw) the highest mean residue levels were also in the blood (122.9 ± 25.98 mg/kg males, 170.9 ± 9.36 mg/kg females) and plasma (100.3 ± 21.06 mg/kg males, 119.7 ± 8.40 mg/kg females). The residue levels in the tissues of females were greater than those of the males.  Overall, the tissue distribution levels present after orally dosing with PC radiolabelled desmedipham were much higher than with EPC radiolabel, reflecting the difference in the rates of excretion. The sex difference of higher tissue residue levels present in the females compared with the males was exhibited by both radiolabelled forms of desmedipham.  The ethyl phenyl carbamate (EPC) radiolabelled dose was more rapidly and completely excreted (>98% of low dose within 30 hours, >99% of high dose within 96 hours) than the phenyl carbamate (PC) radiolabelled dose (>84% of low dose within 48 hours; >91% of high dose within 96 hours).  With both radiolabelled compounds the low dose was excreted more rapidly, showing a dose-related effect.  The major route of excretion was via the urine for both the EPC and PC radiolabelled doses when administered at the low dose level (5 mg/kg bw) (for the EPC radiolabel 89- 95%, and for the PC radiolabel 70-84%). Faecal elimination accounted for a further 5-9% and 13-20% of EPC- and PC-radiolabelled doses, respectively.  When administered as a single oral dose (1000 mg/kg bw) greater proportions of both the EPC- and PC-radlolabelled doses were eliminated via the faeces (51% of the EPC-radiolabel, 56% for the PC radiolabel). Excretion of the EPC-radiolabelled dose was equally divided between urine and faeces but for the PC-radiolabel faecal elimination was the major route with the urine (including the cage wash) accounting for 36-38% of the radioactive dose.  There were no sex differences in the rate or route of excretion of either radiolabelled form of 14C-desmedipham when administered as single oral doses at dose levels of 5 and 1000 mg/kg bw. When administered as a repeat dose (5 mg/kg bw) an increase in the mean percentage of both radiolabelled doses eliminated via the faeces was observed in males dosed with the EPC-radiolabel (80%) and females dosed with the PC-radiolabel (60%). There was a corresponding decrease in the mean percentage excreted via the urine.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-11-06 to 1990-06-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Objective of study:

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

After the study was performed, a new version of the OECD Test Guideline 417 has been adopted 22nd July 2010. Basic TK parameters such as AUC, Cmax, Tmax and plasma half-live are new data requirements which were not calculated.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

Two radiolabelled test materials were used in addition to non-labelled desmedipham

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD Sprague Dawley rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 130- 164g (males) and 133-150g (females).
- Housing: Prior to dosing, animals were housed in plastic holding cages with wood-chip bedding.
After dosing with desmedipham, the five male and five female animals of each treatment group were housed individually in all-glass metabolism cages which allowed free access to food and water, and which were equipped to collect urine and faeces and to trap carbon dioxide in the expired air.
- Diet: Biosure PRD nuts were provided ad libitum except for a period of 16 hours prior to dosing when food was withheld.
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C
- Photoperiod: automatically illuminated with a 12-hour photoperiod of fluorescent lighting.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
PC label
PC radiolabelled desmedipham (6.74 mg) and unlabelled desmedipham (8.54 mg) were dissolved in 15.3 mL polyethylene glycol (approximate molecular weight = 400, PEG 400) to give a dosing solution of specific activity 24.53mCi/g and concentration of 1 mg/mL. Five male and five female rats were orally dosed by intubation with 1ml of the solution/200g bodyweight, thus receiving 5 mg desmedipham/kg bw. A further two animals of each sex were dosed at the same rate with a solution of desmedipham at a specific activity of 59.1mCi/g.

EPC label
EPC radiolabelled desmedipham (6.03 mg) and unlabelled desmedipham (9.9 mg) were dissolved in PEG 400 (16.0 mL) to give a dosing solution of specific activity 23.61 mCi/g and concentration 1 mg/mL. Five male and five female rats were dosed as above. Two further animals of each sex were dosed with a solution of desmedipham at a specific activity of 61.6mCi/g.

Duration and frequency of treatment / exposure:

single oral dose

Dose / conc.:

5 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

2/sex/group

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Urine and faeces were collected from the cages for metabolite analysis. The enzyme hydrolysed urine and faecal samples were analysed by HPLC and TLC.

Metabolites identified:

yes

Details on metabolites:

In rats which received EPC ring radiolabelled desmedipham, the major urinary metabolite was N-(3-hydroxyphenyl) ethyl carbamate (63% of the radioactivity present in urine) with smaller amounts of 3-acetamidophenol (18% of the radioactivity in urine) and 3-aminophenol (4% of the radioactivity in urine). Analysis of faecal homogenates showed the similar pattern of metabolites as in urine, approximately 11% of the administered dose. Analysis of faecal extracts supported the identity of the major metabolites as in faecal homogenates, but the quantitative analysis was not possible due to large amount of co-extracted material. The data on metabolites as percent of the administered dose was not presented in tabular form.

In rats administered PC ring radiolabelled desmedipham, the major metabolite identified in urine was 4- acetamidophenol, with only traces of 4-aminophenol, 2-aminophenol, 2-acetamidophenol and parent desmedipham. Aniline, a proposed intermediate from PC ring part was not detected. In theory, aniline was rapidly and completely converted by hydroxylation in the 4-position to 4-aminophenol and further acetylated to 4-acetamidophenol. All these metabolites found in urine were present as glucuronic acid and/or sulphate conjugates. The major faecal metabolite was 4 -acetamidophenol, with smaller amounts of 4-aminophenol and unchanged desmedipham in rats receiving PC ring radiolabelled desmedipham.

Conclusions:

Desmedipham was rapidly metabolised in the rat via oxidative/hydrolytic cleavage of the parent compound yielding N-(3-hydroxyphenyl) ethyl carbamate from the EPC ring part of the molecule and 4-acetamidophenol from the PC ring part of the molecule. N-(3-hydroxyphenyl) ethyl carbamate was further partially converted into 3-aminophenol with subsequent acetylation to 3-acetamidophenol. Aniline, a proposed intermediate from the PC ring part was not detected. In theory, aniline may convert to 4-aminophenol and further acetylated to 4-acetamidophenol. There were no apparent sex differences in urinary metabolites in rats which received PC or EPC ring radiolabelled test material. There was also present small amounts of polar metabolites, which remained unidentified in urine and faecal homogenates.

Executive summary:

The metabolic fate of the herbicide desmedipham in the rat was investigated following oral administration at 5 mg/kg bw of separate preparations of ¹⁴C-desmedipham radiolabelled in either the phenyl carbamate (PC) or ethyl phenyl carbamate (EPC) ring.  Both radiolabelled forms were completely metabolised and rapidly excreted (>75% in the first 24 hours for PC-labelled material, >90% for the EPC label). The major route of excretion was via the urine which accounted for 56-73% of the dose from animals treated with PC labelled desmedipham and 76-90% following EPC ring labelled treatment.  Tissue residues following administration of EPC-labelled desmedipham were very low, generally below the limit of detection (approximately 0.1 mg/kg depending on tissue). Residues in animals dosed with PC labelled desmedipham were somewhat higher, with highest levels of 0.4-0.8 mg/kg in blood and 0.3-0.8 mg/kg in plasma. Residues in most other tissues were less than 0.25 mg/kg.  The metabolism of desmedipham in the rat involved an initial oxidative/hydrolytic cleavage of the parent compound yielding N-(3-hydroxyphenyl) ethyl carbamate and aniline. The latter compound was rapidly and completely converted to 4-aminophenol, which was in turn largely acetylated to 4-acetamidophenol. The N-(3-hydroxyphenyl) ethyl carbamate was partially converted into 3-aminopheriol with subsequent acetylation to 3-acetamidophenol. All these metabolites were excreted as conjugates.

Reference 6

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993-07-13 to 1994-01-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Objective of study:

tissue distribution

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

After the study was performed, a new version of the OECD Test Guideline 417 has been adopted 22nd July, 2010. AUC is a new data requirement which were not calculated in this study and exhaled air was not investigated.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: 1% gum tragacanth in distilled water

Details on exposure:

Groups of 18 male and 18 female Sprague-Dawley rats were given by gavage a single oral dose of aqueous suspension of PC ring radiolabelled desmedipham at 5 or 1000 mg/kg bw.

Duration and frequency of treatment / exposure:

Single low and high oral dose, orally by gavage

Dose / conc.:

5 mg/kg bw (total dose)

Remarks:

Single oral low dose: PC label

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Single oral high dose: PC label

No. of animals per sex per dose / concentration:

18 animals/sex/dose (3 animals/sex/sacrifice group/dose)

Control animals:

no

Details on study design:

- Dose selection rationale: not specified.
- Rationale for animal assignment (if not random): random.

Details on dosing and sampling:

Sample collection: Low dose; 2, 8, 24, 72, 120, 168 h
High dose; 12, 24, 36, 72, 144, 216 h

Statistics:

The terminal half-life of the radiolabelled desmedipham residues in blood, plasma, liver and kidney were calculated with pharmacokinetics software using the non-compartmental log-linear regression.

Preliminary studies:

In the preliminary study (not available) the blood concentrations were determined in various time points after dosing at the low and high dose levels. The time of peak blood concentrations (Cmax) was found to be approximately 2 and 12 hours for the low and high dose levels, respectively. The decline was slow at both dose levels with blood concentrations still quantifiable 7 days after dosing. In earlier studies in rat, quantifiable residue levels in most tissues were present 4 days after administration of the low dose.

Type:

distribution

Results:

Desmedipham was distributed mainly to organs with a high blood flow e.g., liver, muscles, lungs, kidney, heart, lungs, testes, and spleen. Tissue residue levels were generally slightly higher in females than in males.

Details on distribution in tissues:

At 5 mg/kg bw, all examined tissues contained quantifiable levels of radioactivity. The highest detected concentrations were found in the gastro-intestinal tract and in the liver, kidneys, blood


and plasma 2 hours after dosing. The residues of radioactivity were also detected in the lungs, spleen, thyroids, testes/ovaries, and renal fat 2 hours after dosing. The levels of radioactivity in tissues decreased gradually and 7 days (168 h) after dosing still low levels could be detected.
At 1000 mg/kg bw, in male rats the highest levels of radioactivity were found in the gastro-intestinal tract, blood, plasma and in the liver 12 hours after dosing, when the residues were determined for the first time. The females exhibited higher levels in some tissues (blood, bone, carcass, eyes, kidney, lungs, muscle, ovaries, plasma, thyroids) 24 hours after dosing. After 9 days the levels of residues were reduced, e.g., the liver; 3.6 mg eq/kg in males and 4.3 mg eq/kg in females, but they were still quite high.
Distribution ratio of radiolabelled residues of desmedipham was calculated for all tissues, except bone, using the following formula:

Total dpm present in total weight of tissue or organ/Total dpm administered to rat.

Absolute organ weights were not given in the study report. For blood, plasma, muscle, and fat the total weight of tissue was estimated using values for percentage of total body weight attributable to the appropriate tissue, obtained from published data (blood 7%, plasma 4%, muscle 45.5%, fat 7.1% of total body weight). According to report amendment (31.1.1994) the values of distribution ratios of radiolabelled residues in bone were not calculated, because the figure for the percentage of rat body weight attributable to bone, was not available.
Excluding carcass and G.I tract radiolabelled tissue residues accounted for approximately 0.1 - 6% of the administered dose in blood, plasma, renal fat, liver, and muscle. However, these percentages were based on total weight of the tissue. Radioactivity recovered (percentage of dose) was not presented per gram of tissue in the study report.
Based on the partition coefficient (log Pow 3.39) desmedipham may have had potential to accumulate to a moderate extent. In renal fat radioactivity levels were also notable (0.1 - 1.4% of the dose), but the percentage of the radioactivity decreased with time showing no clear tendency to accumulate.
At both dose levels, clearance of PC ring radiolabelled residues from the tissues of male and female rats occurred at a similar rate.

Key result

Toxicokinetic parameters:

other: Terminal plasma halflife 39.03 - 58.55 hours (PC ring)

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: (low dose) 2.08 mg equivalents [14C]-desmedipham/kg tissue in males and 2.31 mg equivalents [14C]-desmedipham/kg tissue in females

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: (low dose) 2 hours

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: (high dose) 126.12 mg equivalents in males and 134.04 [14C]-desmedipham/kg tissue in females

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: (high dose) 12 hours

At both dose levels, the clearance of radiolabelled residues from tissues occurred at a similar rate, with higher levels of radioactivity in tissues of rats dosed at 1000 mg/kg bw than in rats dosed at 5 mg/kg bw. In blood the values of terminal half-lives and the percentages of the dose were greater than the corresponding plasma values indicating a slower clearance of radiolabelled residues from red blood cells than from plasma. In plasma, the terminal half-lives were shorter (39.0-58.6 hours) than in blood (70-116 hours). Even though the terminal half-lives of the PC ring radiolabelled desmedipham residues were long in blood they were similar at both low and high doses suggesting that elimination of desmedipham did not saturate at high dose levels. In the liver and kidney at dose levels of 5 and 1000 mg/kg bw, the terminal half-lives of PC ring radiolabelled desmedipham residues were of the same order of magnitude (50–84 hours) in males and females. 

Desmedipham was distributed mainly to organs with a high blood flow e.g., liver, muscles, lungs, kidney, heart, lungs, testes, and spleen. By 7 days after the low dose most tissues contained low but quantifiable residues (0.2-0.6 mg eq/kg in blood) and 9 days after a high dose (1000 mg/kg bw) all tissues residue levels were still quite high (28-40 mg eq/kg in blood, 8-7 mg eq/kg in plasma). Detectable residue levels even after 9 days of dosing were compatible with the long elimination half-life of radioactivity in blood. Tissue residue levels were generally slightly higher in females than in males.

 

Tâble 1: Mean concentration (mg equivalents [14C]-desmedipham/kg tissue) of labelled residues in tissues of rats after administration of a single oral dose of PC ring radiolabelled desmedipham at 5 mg/kg bw

Tissue	Single oral dose ^14C-PC-ring 5 mg/kg bw 2 h post dose		Single oral dose ^14C -PC-ring 5 mg/kg bw 24 h post dose		Single oral dose ^14C-PC-ring 5 mg/kg bw 168 h post dose		Terminal half-life (hours) ^14C -PC-ring 5 mg/kg bw
	Male	Female	Male	Female	Male	Female	Male	Female
Blood	2.60	3.71	0.98	1.48	0.24	0.60	70.36	110.42
Plasma	2.08	2.31	0.97	1.23	0.08	0.22	39.03	58.55
Kidney	2.87	3.36	0.37	0.55	0.05	0.15	50.08	73.04
Liver	2.68	4.46	0.28	0.55	0.06	0.10	63.60	59.43
Lungs	0.90	1.35	0.33	0.49	0.07	0.19
Carcass	0.66	0.90	0.24	0.33	0.07	0.15
G.I. tract	15.97	22.54	0.19	0.46	0.02	0.04
Brain	0.19	0.33	0.03	0.05	BLQ	0.02
Renal fat	1.02	0.97	0.19	0.25	0.02	0.05
Bone	0.42	0.55	0.11	0.19	0.03	0.06
Adrenals	0.57	1.02	0.17	0.26	0.06	0.12
Spleen	0.69	1.13	0.20	0.37	0.06	0.15
Heart	0.86	1.01	0.27	0.39	0.06	0.12
Thyroid	0.49	0.57	0.21	0.25	0.08	0.18
Ovaries		0.82		0.31		0.10
Testes	0.45		0.15		0.01
Eyes	0.18	0.21	0.05	0.06	0.02	0.04

Table 2: Mean concentration (mg equivalents [14C]-desmedipham/kg tissue) of labelled residues in tissues of rats after administration of a single oral PC- ring radiolabelled desmedipham at 1000 mg/kg bw

Tissue	Single oral dose ^14C-PC-ring 1000 mg/kg bw 12 h post dose		Single oral dose ^14C -PC-ring 1000 mg/kg bw 72 h post dose		Single oral dose ^14C -PC-ring 1000 mg/kg bw 216 h post dose		Terminal half-life (hours) ^14C -PC-ring 1000 mg/kg bw
	Male	Female	Male	Female	Male	Female	Male	Female
Blood	165.92	218.44	69.84	95.90	28.89	40.62	95.68	116.19
Plasma	126.12	134.04	61.40	69.39	8.33	7.06	48.33	40.49
Kidney	75.47	86.82	24.09	34.07	7.52	8.52	84.17	66.31
Liver	95.72	82.34	13.58	15.95	3.58	4.30	69.69	76.14
Lungs	59.10	72.67	25.50	28.42	9.13	11.98
Carcass	35.51	54.69	19.66	24.45	9.39	9.30
G.I. tract	1519.5	2321.2	9.32	8.53	2.86	2.48
Brain	13.68	15.79	3.34	4.51	1.60	2.04
Renal fat	55.34	46.27	8.86	9.24	3.05	2.04
Bone	24.39	27.33	8.87	11.73	4.44	3.83
Adrenals	34.32	48.30	7.66	14.61	3.29	4.13
Spleen	43.45	52.69	13.28	20.27	5.37	5.29
Heart	50.30	63.22	17.83	22.99	6.96	10.11
Thyroid	27.74	30.49	13.31	13.13	7.64	5.64
Ovaries		47.43		14.71		4.60

Testes	23.49		10.03		1.85
Eyes	10.38	10.76	4.75	5.90	3.56	3.02

 

Table 3: Mean distribution ratios of radiolabelled residues in tissues of rats following administration of a single dose of PC-ring radiolabelled desmedipham at 5 mg/kg bw

Tissue/ Organ		Distribution	ratio x 100
	5 mg/kg 2 h male female		5 mg/kg 8 h male    female		5 mg/kg 24 h male	female	5 mg/kg 72 h male female		5 mg/kg 168 h male female
Blood	3.6	5.1	2.3	4.1	1.3	2.0	0.9	1.1	0.3	0.8
Plasma	1.6	1.8	1.3	1.8	0.8	1.0	0.4	0.4	0.05	0.2
Muscle	3.2	6.3	1.8	2.7	1.2	1.7	0.9	1.1	0.3	0.7
Renal Fat	1.4	1.3	0.6	0.7	0.3	0.4	0.1	0.1	0.03	0.06

 

Taqble 4: Mean distribution ratios of radiolabelled residues in tissues of rats treated with a single dose of PC-ring radiolabelled desmedipham at 1000 mg/kg bw

Tissue/ Organ		Distribution	ratio x 100
	1000 mg/kg 12 h male female		1000 mg/kg 24 h male    female	1000 mg/kg 36 h male      female		1000 mg/kg 72 h male female		1000 mg/kg 216 h male female
Blood	1.2              1.5		0.8            1.8	0.7	1.2	0.5	0.7	0.2	0.3
Plasma	0.5              0.5		0.5            0.8	0.4	0.6	0.2	0.3	0.03	0.03
Muscle	1.2              1.2		0.8            1.3	0.9	1.1	0.5	0.6	0.2	0.2
Renal Fat	0.4              0.3		0.1           0.3	0.1	0.2	0.06	0.06	0.02	0.01

 

Table 5: Distribution ratios of radiolabelled residues in some tissues of rats 2, 12 and 24 hours after administration of a single oral dose of PC-ring radiolabelled desmedipham at 5 and 1000 mg/kg bw

Tissue/Organ	Distribution ratio x 100
	5 mg/kg 2 h male	female	5 mg/kg 24 h male	female	1000 mg/kg 12 h male          female		1000 mg/kg 24 h male       female
Liver	1.8	3.2	0.4	0.6	0.5	0.4	0.2	0.5
Kidney	0.5	0.6	0.1	0.1	0.1	0.1	0.04	0.1
Lungs	0.1	0.2	0.04	0.06	0.04	0.05	0.03	0.06
Testes/Ovaries	0.1	0.02	0.04	0.005	0.03	0.004	0.02	0.004
Spleen	0.04	0.06	0.01	0.02	0.01	0.02	0.006	0.02
Bone	NA	NA	NA	NA	NA	NA	NA	NA
Brain	0.04	0.07	0.006	0.01	0.01	0.02	0.005	0.01
Heart	0.07	0.09	0.02	0.04	0.02	0.03	0.02	0.03
Carcass	9.4	2.9	3.7	5.2	2.6	4.1	2.4	5.0
G.I tract	45.2	55.6	0.6	1.3	24.4	32.4	2.9	12.7

Conclusions:

Desmedipham was distributed mainly to organs with a high blood flow e.g., liver, muscles, lungs, kidney, heart, lungs, testes, and spleen. Tissue residue levels were generally slightly higher in females than in males. Terminal plasma half-life was calculated as 39.03-58.55 hours (PC ring) low dose (5 mg/kg bw):
Cmax: 2.08 mg equivalents [14C]-desmedipham/kg tissue in males and 2.31 mg equivalents [14C]-desmedipham/kg tissue in females
Tmax: 2 hours
High dose (1000 mg/kg bw:
Cmax: 126.12 mg equivalents in males and 134.04 [14C]-desmedipham/kg tissue in females
Tmax: 12 hours
Desmedipham was distributed mainly to organs with a high blood flow e.g., liver, muscles, lungs, kidney, heart, lungs, testes, and spleen. Tissue residue levels were generally slightly higher in females than in males.
Terminal plasma halflife 39.03-58.55 hours (PC ring) low dose (5 mg/kg bw):
Cmax: 2.08 mg equivalents [14C]-desmedipham/kg tissue in males and 2.31 mg equivalents [14C]-desmedipham/kg tissue in females
Tmax: 2 hours
High dose (1000 mg/kg bw/day):
Cmax: 126.12 mg equivalents in males and 134.04 [14C]-desmedipham/kg tissue in females
Tmax: 12 hours.

Executive summary:

Following the OECD guideline of TG 417, groups of 18 male and 18 female Sprague-Dawley rats were given by gavage a single oral dose of aqueous suspension of PC ring radiolabelled desmedipham at 5 or 1000 mg/kg bw. Three male and three female rats from the low dose group were killed at 2, 8, 24, 72, 120 and 168 hours and from the high dose group at 12, 24, 36, 72, 144 and 216 hours post dosing. The tissue concentrations and distribution ratios of radiolabelled residues were determined. Total radioactivity and tissue residues were analysed by liquid scintillation counting. The terminal half-life of the radiolabelled desmedipham residues in blood, plasma, liver, and kidney were calculated with pharmacokinetics software using the non-compartmental log-linear regression.  Desmedipham was distributed mainly to organs with a high blood flow e.g., liver, muscles, lungs, kidney, heart, lungs, testes, and spleen. Tissue residue levels were generally slightly higher in females than in males.

Terminal plasma halflife 39.03-58.55 hours (PC ring) low dose (5 mg/kg bw):
Cmax: 2.08 mg equivalents [14C]-desmedipham/kg tissue in males and 2.31 mg equivalents [14C]-desmedipham/kg tissue in females
Tmax: 2 hours
High dose (1000 mg/kg bw):
Cmax: 126.12 mg equivalents in males and 134.04 [14C]-desmedipham/kg tissue in females
Tmax: 12 hours
Desmedipham was distributed mainly to organs with a high blood flow e.g. liver, muscles, lungs, kidney, heart, lungs, testes and spleen. Tissue residue levels were generally slightly higher in females than in males.
Terminal plasma halflife 39.03-58.55 hours (PC ring) low dose (5 mg/kg bw):
Cmax: 2.08 mg equivalents [14C]-desmedipham/kg tissue in males and 2.31 mg equivalents [14C]-desmedipham/kg tissue in females
Tmax: 2 hours
High dose (1000 mg/kg bw/day):
Cmax: 126.12 mg equivalents in males and 134.04 [14C]-desmedipham/kg tissue in females
Tmax: 12 hours.

Description of key information

Studies of toxicokinetics, metabolism and distribution following oral administration in the rat are available for ¹⁴C-radiolabelled desmedipham.

The absorption of desmedipham was found to be relatively fast and extensive (about 80% in 24 hours).  Desmedipham is widely distributed with higher amounts of radioactivity identified in blood, plasma, liver, lungs, kidneys, heart, spleen, ovaries, testes, thyroids and adrenals.  In general, tissues radioactivity levels in females were higher than those observed in males.  Around 90% of desmedipham is excreted within 24 hours of administration, mainly via the urine.  Desmedipham is rapidly metabolised in the rat via oxidative/hydrolytic cleavage of the parent molecule; hydroxylation of aromatic rings; acetylation of amine groups; and conjugation.  Unchanged desmedipham was only observed in faeces after high administration of high doses.  In a comparative interspecies (rat and human) metabolism study in vitro, no significant quantitative differences and no human-specific metabolites were observed.

Based on the extensive metabolism of desmedipham and the rapid excretionof its metabolites, it can be concluded that there is no potential for bioaccumulation.

Test method/  species	Result	Assessment	Reference
OECD 417 - ADME study in the rat, using a mixture of labelled forms.  Single low and high dose levels of 1 and 100 mg/kg bw.	Desmedipham was moderately absorbed, with urine as the major route of excretion.  Metabolism was extensive and primarily by hydrolysis.  Excretion was rapid and complete; tissue concentrations at 7 days were low and werehighest in whole blood.  Blood residues at the high dose level were disproportionately high.	Supporting study	Prout et al (1995)
OECD 417 - Metabolism study in the rat with two different labelled forms of desmedipham.  Single low dose level 5 mg/kg bw.	Absorption was calculated to be 79-86% based on urinary excretion of radioactivity in 96 hours; the majority of excretion occurred within 24 hours.  Tissue distribution was wide, with highest residues detected in the blood (generally higher in females, and for the PC label compared to the EPC label site).  Metabolites were identified as N-hydroxyphenylethyl carbamate, 3-acetamidophenol and 3-aminophenol (EPC-labelled desmedipham); and (PC-labelled desmedipham); 4-acetamidophenol, 4-aminophenol, 2-aminophenol and 2-aectamidophenol.  Aniline was not detected, but was postulated as an intermediate metabolite.	Supporting study	Challis et al (1990)
OECD 417 - TK study in the rat with two different labelled forms of desmedipham.  Single and multiple low dose (5 mg/kg bw) and single high dose (1000 mg/kg bw).	Absorption at the low dose level was 63-83% and was unaffected by repeated exposure.  Absorption was lower (33-43%) at the high dose level.  Tissue residue levels were generally low, but highest in the blood and plasma and were markedly higher with the PC label compared to the EPC label, and were slightly higher in females compared to males.	Supporting study	Creedy (1993)
OECD 417 - Clearance study in the rat following single low (5 mg/kg bw) or high dose (1000 mg/kg bw) of desmedipham (PC-ring labelled)	Desmedipham was distributed mainly to well-perfused organs; tissue residues were generally higher in females.  Terminal plasma half-life was calculated as 39-59 hours.  Tmax was 2 hours for the low dose, 12 hours for the high dose	Supporting study	Creedy (1993)
OECD 417 - Metabolism study in the rat with two different labelled forms of desmedipham.  Single or repeated low dose level 5 mg/kg bw; single high dose level 1000 mg/kg bw.	Faecal radioactivity was higher at the high dose level, consistent with reduced absorption.  For EPC-labelled desmedipham, the major metabolite was identified as N-hydroxyphenyl ethyl carbamate (conjugates in urine, free in faeces).  3-aminopehnol and acetamidophenol were also detected, mainly as conjugates.   For PC-labelled desmedipham, the major faecal metabolite was phenylmethyl carbamate; the major urinary metabolite was 4-acetamidophenol.	Supporting study	Jackson (1993)
None available - Comparative in vitro metabolism study in rat and human hepatocytes	The study did not identify any unique human metabolites	Supporting study	Sola (2015)
None available - QWBA study in CD-1 mice to support genetox studies	The study demonstrates exposure of the bone marrow in male CD-1 mice administered a single oral dose of 2000 mg/kg bw radiolabelled desmedipham.	Supporting study	Sandmann (2017)

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

80

Absorption rate - dermal (%):

8

Absorption rate - inhalation (%):

100