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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 January 2014 to 10 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline 417 compliant

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
certificate no. 2012/16

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate
EC Number:
700-204-6
Cas Number:
1174627-68-9
Molecular formula:
C9H17NO3
IUPAC Name:
methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Rhodiasolv Polarclean
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Italy
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 252 to 306 g
- Fasting period before study: no
- Housing: During the pre-treatment period and for animals treated for blood pharmacokinetics and tissue distribution, the animals were housed by four from the same group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2, 61 cm x 43.5 cm x 21.5 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained enrichment (rat cocoon).
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 5059852 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 8 January 2014 To: 10 February 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
[14C]-Rhodiasolv Polarclean was diluted as appropriate with corresponding non-radiolabelled compound so that the animals received approximately 2.2 MBq/kg by administration.
The dose formulation was prepared as follows:
 on the treatment day, the required quantity of Rhodiasolv Polarclean was added in a solution of [14C] Rhodiasolv Polarclean in drinking water by reverse osmosis in gauged preparation vial,
 the solution was added in the final required quantities of vehicle, then the solution was aliquoted and kept at room temperature until use.

The radiolabelled test item and the isotopic dose formulation were analysed as follows:
- Radiolabelled test item:
 the total radioactivity of the radiolabelled test item solution (MBq/mL of radiolabelled test item solution) was determined the day before dosing or the day of dosing on three aliquots (by weight) using the Tri-carb 2910TR Liquid Scintillation system (Perkin Elmer, Downers Grove IL 60515, USA),
 the radiochemical purity of the radiolabelled test item was controlled in duplicate using a HPLC/UV/on line radioactivity detection, the day before the radiolabelled treatment or the day of the radiolabelled treatment. The radiochemical purity was > 95%.

- Isotopic dose formulation:
 the total radioactivity of the dose formulation was determined on the day of dosing on three aliquots (by weight) using the Tri-carb 2910TR Liquid Scintillation system (Perkin Elmer, Downers Grove IL 60515, USA). After treatment, the same analysis of radioactivity was undertaken on the three aliquots of the dose formulations which have passed through the same administration devices as that used for the treatment,
 the radiochemical purity of the dose formulation was determined in duplicate using a HPLC/UV/on line radioactivity detection on the day of radiolabelled dosing. The radiochemical purity of the dose formulation was > 95%


VEHICLE
- Concentration in vehicle: 200 and 400 mg/mL
- Amount of vehicle (if gavage):

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Duration and frequency of treatment / exposure:
Each animal was treated by oral (gavage) administration on a single occasion (Day 1).
Doses / concentrations
Remarks:
Doses / Concentrations:
- Group 1 (Pharmacokinetics): 2.2 MBq/kg and 1000 mg/kg bw
- Group 2 (Pharmacokinetics): 2.2 MBq/kg and 2000 mg/kg bw
- Group 3 (Mass Balance/Tissue Distribution): 2.2 MBq/kg and 2000 mg/kg bw
- Group 4 (Tissue Distribution): 2.2 MBq/kg and 2000 mg/kg bw
No. of animals per sex per dose / concentration:
12 males for Groups 1 and 2,
4 males for Groups 3 and 4.
Control animals:
no
Positive control reference chemical:
no
Details on study design:
- Dose selection rationale: The dose-levels were selected based on the results of an acute oral (gavage) toxicity study (Nagy, 2009) where the test item induce no mortality and no clinical signs in rats treated at the dose of 2000 mg/kg.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, plasma
- Time and frequency of sampling: Blood samples were taken from animals of Groups 1 and 2 as follows:
 first set of four animals: 0.25, 2 and 8 hours post-dosing,
 second set of four animals: 0.5, 3 and 24 hours post-dosing,
 third set of four animals: 1, 4 and 48 hours post-dosing.

MASS BALANCE (MB) AND TISSUE DISTRIBUTION (TD):
- Blood collection (Groups 3 and 4; MB and TD):
Blood samples were taken from animals of Groups 3 and 4 as follows:
 0.25 hours corresponding to Tmax determined during the PK phase for Group 4,
 168 hours post-dosing for Group 3.

- Excreta collection (Group 3; MB):
Urine and feces were collected from Group 3 animals at the following times:
 over a period of 24 hours before the first dosing (during period -96/-72h) and then during the periods 0-6, 6-24, 24-48, 48 72, 72 96, 96-120, 120-144 and 144 168 hours post-dosing.

- Tissue distribution (Groups 3 and 4, TD)
Liver, fat, gastrointestinal tracts (stomach, duodenum, ileum, jejunum, cecum, colon, gastrointestinal contents and rectum), kidney, spleen and residual carcass were collected from animals of Groups 3 and 4 at the following times:
 0.25 hours corresponding to Tmax determined during the PK phase for Group 4,
 168 hours post-dosing for Group 3

- Method type(s) for identification: Liquid scintillation counting. In addition, selected samples (plasma and urine samples), which have adequate levels of radioactivity , were analyzed for metabolic pattern, by radio HPLC after the appropriate preparation.
- Limits of detection and quantification: limit of detection = 20 dpm/mL for HPLC. Limit of quantification depends on the biological matrices and tissue (ranging from 0.42 Bq/g for urine up to 124 Bq/g for feces).
- Other:
Statistics:
no performed.

Results and discussion

Preliminary studies:
Not applicable.
Main ADME resultsopen allclose all
Type:
absorption
Results:
The test item was almost entirely absorbed, with percentage of absorption of 100.1%
Type:
distribution
Results:
The radioactivity was mainly observed in the gastrointestinal tract 0.25 hours after oral administration and distributed in the other tissues (liver, spleen, kidney and fat) at low concentration levels.
Type:
metabolism
Results:
Two metabolites without the presence of the parent drug were found in urine and only one metabolite in plasma. The main metabolite in urine was also seen in plasma. This suggests a complete metabolization of the test item.
Type:
excretion
Results:
The overall elimination of the radioactivity occurred within 24 hours after administration and mainly in urine. Only 1.2% of the dose was found in feces. Recovery was complete at 101.2%.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Mean total radioactivity levels were quantifiable in plasma from the first time-point of 0.25 to 8 hours for both dose-levels (1000 and 2000 mg/kg). The maximum mean plasma total radioactivity was reached at 0.25 hours after oral administration. Then the mean total radioactivity had tendency to decrease until 8 hours in a multi-compartmental manner.
Following single administration at a dose-level of 2000 mg/kg and 2.2 MBq/kg, the mean cumulative percentage of absorption, over a 168-hour period, was 100.1% of the dose administered (see table 7.1.1/3).
Details on distribution in tissues:
Following single oral (gavage) administration, the radioactivity levels were quantifiable at 0.25 hours (corresponding to the Tmax) in all tissues. Specifically, the radioactivity was mainly in the gastrointestinal tracts, with 91.8% of dose administered and content was 13821 µg.eq/g. In other tissues (liver, spleen, fat and kidney), the radioactivity levels were below than 2% of the administered dose and contents were 105 2178 µg.eq/g. In contrast, 168 hours after treatment, the radioactivity levels were below the Limit Of Quantification in all tissues suggesting a total elimination of the test item 168 hours after treatment (see Table 7.1.1/4).
Details on excretion:
The radioactivity was eliminated mainly in the urine. Specifically, 88.3% of the dose over the study period was measured in the urine versus 1.2% in the feces.
The administered radioactivity was excreted rapidly, as 87.0% of the dose in urine (equivalent to > 98% of the total recovered dose in urine) was eliminated within the first 24 hours after administration. Thereafter, the elimination was less than 0.8% of the dose par day with a decreasing trend over time.

For the feces, most of radioactive dose (0.94% of the dose, equivalent to > 81% of the total recovered dose in feces) was eliminated rapidly within the first 24 hours after administration. Then, 0.21% of the radioactive dose was eliminated in the feces fraction 24-48 hours, after which elimination was not quantifiable.

Over the study, 11.7% of the dose was recovered in the cage wash. Specifically, 10.4% of the dose in cage wash (equivalent to 88.8% of the total dose recovered in the cage wash) was recovered in cage wash fraction 0-6 hours, after the recovery it was less than 0.8% of the dose per day (see table 7.1.1/2).
Toxicokinetic parametersopen allclose all
Toxicokinetic parameters:
half-life 1st: 5.3 at 1000 mg/kg bw and 4.0 hours at 2000 mg/kg bw
Toxicokinetic parameters:
AUC: 530 µg.eq/g at 1000 mg/kg bw and 812 µg.eq/g at 2000 mg/kg bw
Toxicokinetic parameters:
Cmax: 743 µg.eq.h/g at 1000 mg/kg bw and 1686 µg.eq.h/g at 2000 mg/kg bw
Toxicokinetic parameters:
Tmax: 0.25 h at 1000 and 2000 mg/kg bw

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Metabolic pattern analysis in plasma samples at the time-points 0.25 and 0.5 hours showed the presence of one metabolite without presence of the parent drug. Moreover, metabolic pattern analysis in urine fraction 0-6 and 6-24 hours showed the presence of two metabolites without presence of the parent drug. The main metabolite in urine was also seen in plasma. In conclusion, the Rhodiasolv Polarclean was probably entirely metabolized.

Any other information on results incl. tables

Mortality/morbidity and clinical signs:

No unscheduled mortality or morbidity and clinical signs were noted during the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Following single oral (gavage) administration of the isotopic Rhodiasolv Polarclean mixture at dose-levels of 1000 and 2000 mg/kg, and 2.2 MBq/kq of [14C]-Rhodiasolv Polarclean to male Sprague-Dawley rats, the test item was well tolerated. The maximum plasma concentration was reached 0.25 hours post-dosing for both dose-levels and the terminal half-life values were similar. The plasma exposure increased dose proportionally between 1000 and 2000 mg/kg.
The test item was almost entirely absorbed, with percentage of absorption of 100.1%. The overall elimination of the radioactivity occurred within 24 hours after administration and mainly in urine. Only 1.2% of the dose was found in feces. Recovery was complete at 101.2%.
The radioactivity was mainly observed in the gastrointestinal tract 0.25 hours after oral administration and distributed in the other tissues (liver, spleen, kidney and fat) at low concentration levels. Nevertheless, at 168 hours post dosing, the radioactivity levels in all tissues were below the Limit Of Quantification, confirming the complete elimination of the test item.
Two metabolites without the presence of the parent drug were found in urine and only one metabolite in plasma. The main metabolite in urine was also seen in plasma. This suggests a complete metabolization of the test item.
Executive summary:

The objective of this ADME study, performed according to OECD 417 guideline and in compliance with GLP, was to investigate the blood and plasma pharmacokinetics, mass balance and tissue distribution of total radioactivity following a single oral (gavage) administration ofradiolabelled test item, [14C]-Rhodiasolv Polarclean,in male Sprague-Dawley rats. In addition the presence of metabolites in plasma and urine was investigated.

Method:

Thirty two Sprague-Dawley male rats were allocated to four groups and were orally (gavage) treated on a single occasion (Day 1), as follows:

. Group 1, twelve rats treated at the dose-levels of 2.2 MBq/kg and 1000 mg/kg for Pharmacokinetic evaluation,

. Group 2, twelve rats treated at the dose-levels of 2.2 MBq/kg and 2000 mg/kg for Pharmacokinetic evaluation,

. Group 3, four rats treated at the dose-levels of 2.2 MBq/kg and 2000 mg/kg for evaluation of the mass balance and tissue distribution,

. Group 4, four rats treated at the dose-levels of 2.2 MBq/kg and 2000 mg/kg for evaluation of the tissue distribution. 

A constant dosage volume of 5 mL/kg was used for the oral administration. The isotopic test item mixture was given as a solution in drinking water treated by reverse osmosis.

Blood samples were collected in Groups 1 and nine time-points over a 48-hour period. Blood samples were collected in Groups 3 and 4 just before sacrifice. Excreta were collected in Groupfractions of 6, 18 or 24 hours, pre-dose, and then over a 168-hour period. Various tissues and organs were taken at sacrifice in Groups 3 (at 168 hours post-dosing) and 4 (at 0.25 hours post-dosing, corresponding to the Tmax).

Biological samples were analysed for total radioactivity by liquid scintillation counting. Selected plasma and urine samples were analysed for the metabolic pattern. No metabolic pattern was performed forfecesas the radioactivity levels were too low.

Mortality, morbidity andclinical signswere checked at least once a day. The body weight of each animal was recorded once before group allocation, then on the day of treatment for all groups and before sacrifice for Groups 3 and 4. After the final blood sampling, the animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by cervical dislocation.

 

Results:

Achieved dosage form:

There was a good agreement between the nominal and actual dose-levels calculated for all dose formulations, as all deviations were within ± 5% of the target values. There was a satisfactory agreement between the nominal and actual total radioactivity found in all dose formulations, with deviations within ± 20%.

Pharmacokinetic analysis:

After a single oral (gavage) administration at the dose-levels of 1000 and 2000 mg/kg of isotopic test item mixture and at 2.2 MBq/kg of [14C]-Rhodiasolv Polarclean, a low to moderate inter-animal variability was observed on total radioactivity concentrations in plasma. A moderate inter-animal variability was observed on total radioactivity concentrations in blood.

 

Mean total radioactivity levels were quantifiable in plasma from the first time-point of 0.25 to 8 hours for both dose-levels. The maximum mean plasma total radioactivity was reached 0.25 hours after oral administration. Then the mean total radioactivity decreased until 8 hours in a multi‑compartmental manner.  

Plasma total radioactivity pharmacokinetic parametersfollowing a single oral (gavage) administration at the dose-levels of 1000 and 2000 mg/kg of isotopic test item mixture and at 2.2 MBq/kg of [14C]‑Rhodiasolv Polarclean to male Sprague-Dawley rats are presented in the table below:

 

Group

1

2

Dose-level (in mg/kg and in MBq/kg)

1000 and 2.2

2000 and 2.2

Tmax

h

0.25

0.25

Cmax

µg.eq/g

530

812

AUC0-t

µg.eq.h/g

743

1686

λz

1/h

0.131

0.174

t1/2

h

5.3

4.0

Cmax/ Dose

0.530

0.406

AUC0-t/ Dose

0.743

0.843

 

The terminal half-life values were similar. The plasma exposure based on Cmaxand AUC0-thad tendency to increase dose-proportionally between 1000 and 2000 mg/kg.

Mass Balance:

Following single oral (gavage) administration, the mean total cumulative excretion in urine, feces and cage wash of the administered radioactive dose, over a 168-hour period, was 101.2%.

The radioactivity was eliminated mainly in the urine. Specifically, 88.3% of the dose over the study period was measured in the urineversus1.2% in the feces.

The administered radioactivity was excreted rapidly (within the first 24 hours after administration). For the feces, most of radioactive dose was also eliminated rapidly (within the first 24 hours after administration).

Following single oral (gavage) administration at a dose-level of 2000 mg/kg and 2.2 MBq/kg to male Sprague-Dawley rats, the mean cumulative percentage of absorption, over a 168-hour period, was 100.1% of the dose administered.

Tissue distribution:

Following single oral (gavage) administration, the radioactivity levels were quantifiable at 0.25 hours (corresponding to the Tmax) in all tissues. Specifically, the radioactivity was mainly in the gastrointestinal tracts, with 91.8% of dose administered. In other tissues (liver, spleen, fat and kidney), the radioactivity levels were below than 2% of the administered dose. In contrast, 168 hours after treatment, the radioactivity levels were below the limit of quantification in all tissues. Metabolic pattern:

Metabolic pattern analysis in plasma samples at the time-points 0.25 and 0.5 hours showed the presence of one metabolite without presence of the parent drug. Moreover, metabolic pattern analysis in urine fraction 0-6 and 6-24 hours showed the presence of two metabolites without presence of the parent drug. The main metabolite in urine was also seen in plasma.

 

Mortality/morbidity and clinical signs

No unscheduled mortality or morbidity and clinical signs were noted during the study.

 

Conclusion

 Following single oral (gavage) administration of the isotopic Rhodiasolv Polarclean mixture at dose-levels of 1000 and 2000 mg/kg, and 2.2 MBq/kq of [14C]-Rhodiasolv Polarclean to male Sprague-Dawley rats, the test item was well tolerated. The maximum plasma concentration was reached 0.25 hours post-dosing for both dose-levels and the terminal half-life values were similar. The plasma exposure increased dose‑proportionally between 1000 and 2000 mg/kg.

The test item was almost entirely absorbed, with percentage of absorption of 100.1%. The overall elimination of the radioactivity occurred within 24 hours after administration and mainly in urine. Only 1.2% of the dose was found in feces. Recovery was complete at 101.2%.

The radioactivity was mainly observed in the gastrointestinal tract 0.25 hours after oral administration and distributed in the other tissues (liver, spleen, kidney and fat) at low concentration levels. Nevertheless, at 168 hours post‑dosing, the radioactivity levels in all tissues were below the Limit Of Quantification, confirming the complete elimination of the test item.

Two metabolites without the presence of the parent drug were found in urine and only one metabolite in plasma. The main metabolite in urine was also seen in plasma. This suggests a complete metabolization of the test item.