Potassium carbamoylcarbamate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-24 to 2012-10-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to an accepted protocol.

Objective of study:

metabolism

Principles of method if other than guideline:

The purpose of this study was to determine the formation of urea after incubation of potassium allophonate in rat liver microsomes, with a secondary objective of evaluating the stability of potassium allophonate under the incubation conditions. Potassium allophonate at a concentration of 10 μM was incubated at 37 deg C in rat liver microsomes for 0, 10, 20, 30 and 60 minutes, in the presence and absence of reduced nicotinamide adenine dinucleotide phosphate (NADPH).

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

not applicable (in vitro study)

Route of administration:

other: not applicable (in vitro study)

Vehicle:

other: not applicable (in vitro study)

Details on exposure:

A 1 mM stock solution of potassium allophonate in 50/50 methanol/water was diluted in microsomal incubation buffer to a final concentration of 10 µM potassium allophonate. The stock solutions used for microsomal incubations were always freshly prepared and used on the same day. The 10 µM potassium allophonate solution was incubated (in triplicate) both in the presence and absence of a reduced nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system (3.6 mM glucose-6-phosphate [G6P], 0.4 U/mL glucose-6-phosphate dehydrogenase [G6PD], 1.3 mM nicotinamide adenine dinucleotide phosphate [NADP+]) for 60 min. The rat microsomal fraction was added to the test system immediately after the time zero sampling event (which actually took place 6 seconds after incubation).

The initial results from these experiments did not achieve the primary objective of this study (to study potassium allophonate conversion to urea in the presence of rat liver microsomes) in that 100% conversion to urea was observed at time 0 (i.e., prior to incubation with rat microsomal fraction), suggesting that potassium allophonate may be converted to urea prior to incubation in rat liver microsomes. To test this hypothesis, the stability of potassium allophonate and formation of urea was investigated in dimethyl sulfoxide (DMSO, CAS No. 67-68-5)/water at the following ratios: 5/95, 10/90, 20/80, 30/70, 40/60 (DMSO/water) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Duration and frequency of treatment / exposure:

0, 10, 20, 30 and 60 min

Remarks:

Doses / Concentrations:
10 µM

No. of animals per sex per dose / concentration:

not applicable

Control animals:

other: not applicable (in vitro study)

Positive control reference chemical:

Midazolam (CAS No. 59467-70-8) and diclofenac (CAS No. 15307-86-5) served as the positive controls and were tested under the same test system as potassium allophonate.

Details on dosing and sampling:

Sample aliquots were removed for analysis at 0, 10, 20, 30 and 60 minutes, and stopped by the addition of acetonitrile (CAS No. 75-05-8) containing 0.2 µM dextrorphan (CAS No. 125-73-5) as an internal standard. Samples were centrifuged at 3400 rpm for 10 min to pellet the precipitated proteins. The supernatant was evaporated to dryness using a Turbovap and aliquots of the resuspended sample used for analysis for the formation of urea and amount of potassium allophonate remaining. Similar incubations were performed using positive control compounds midazolam and diclofenac to confirm the metabolic activity of the microsomes.

Type:

other: hydrolysis

Results:

100% conversion to urea was observed prior to addition of the S9 rat microsomal fraction, suggesting conversion of potassium allophonate to urea prior to incubation in rat liver microsomes

Metabolites identified:

yes

Details on metabolites:

The percent of potassium allophonate remaining in the presence and absence of NADPH was 0% at all time points. After incubation for 0, 10, 20, 30 and 60 minutes in the absence of NADPH, the percent of urea formed was 100%, 101%, 103%, 76% and 78%, respectively. In the presence of NADPH, the percent of urea formed was 100%, 90%, 72%, 69% and 69%, respectively.

The positive control compounds midazolam and diclofenac were metabolized in rat liver microsomes as expected. After incubation for 0, 10, 20, 30 and 60 minutes in the presence of NADPH, the percent of midazolam remaining was 100%, 58%, 27%, 17% and 0.31%, respectively. The percent of diclofenac remaining after 0, 10, 20, 30 and 60 minutes in the presence of NADPH was 100%, 91%, 83%, 73% and 54%, respectively. Midazolam was highly and diclofenac was moderately cleared in rat liver microsomes. These data confirm the metabolic activity of rat liver microsomes used in this study.

Conclusions:

Interpretation of results (migrated information): other: Potassium allophonate was hydrolyzed to urea prior to addition of the rat S9 microsomal fraction (indicating it is susceptible to hydrolysis)
The stability of potassium allophonate was investigated when tested with rat S9 microsomal fraction both in the presence and absence of NADPH. The time zero sampling event showed that all potassium allophonate was converted to urea in both test systems, suggesting that conversion of potassium allophonate occurs prior to incubation in rat liver microsomes (i.e., occurs via hydrolysis). To test this, the stability of potassium allophonate in the DMSO/water was investigated. The results from this test showed that potassium allophonate was converted to urea in all combinations of DMSO/water. Based on these data, it was concluded that potassium allophonate is unstable in DMSO/water, and is hydrolysed to urea when formulated in this vehicle.

Description of key information

An in vitro metabolism study, the stability of potassium allophonate was investigated when tested with rat S9 microsomal fraction both in the presence and absence of NADPH. Under both test conditions, complete conversion to urea was observed at time zero, indicating that conversion of potassium allophonate occurs prior to incubation in rat liver microsomes (i.e., occurs via hydrolysis). Based on the results of this test, it is clear that potassium allophonate has no potential to bioaccumulate in the human body.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

An in vitro metabolism study is available for potassium allophonate, the formation of urea after incubation of potassium allophonate in S9 rat liver microsomes was investigated (Ricerca Biosciences, 2013). Potassium allophonate was tested at a concentration of 10 μM and was incubated at 37 deg C in rat liver microsomes for 0, 10, 20, 30 and 60 minutes, in the presence and absence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The time zero sampling event (prior to addition of S9) showed that all potassium allophonate was converted to urea in both test systems, suggesting that conversion of potassium allophonate occurs prior to incubation in rat liver microsomes (i.e., occurs via hydrolysis). This was confirmed in stability tests using different ratios of the study vehicle, dimethyl sulfoxide (DMSO)/water. Based on the results of this test, it is clear that potassium allophonate has no potential to bioaccumulate in the human body.

References:

Ricerca Biosciences, LLC. 2013. Metabolic Stability of Potassium Allophonate and Formation of Urea in Rat Liver Microsomes. Testing laboratory: Ricerca Biosciences, LLC, Drug Safety and Metabolism, 7528 Auburn Road, Concord OH 44077. Report no.: 029722. Report date: 2013-03-01.