1-butylpyrrolidin-2-one

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-19 to 2016-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

excretion

metabolism

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

deviations from the protocol did not impact the quality or integrity of the study.

GLP compliance:

yes

Remarks:

in compliance with the United States FDA GLP Regulations (21 CFR Part 58), 05 Oct 1987.

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.7%
- Specific activity: 1 x2.0 mCi
- Locations of the label: The test article was [14C]-labeled in the 2'-position of the benzene ring (C=O) (please see picture below).

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

In general, this species and breed of animal is recognized to be appropriate for this type of metabolism study. The rat was utilized because it is a widely used species for which significant historical control data are available, and because it has been determined to be a pharmacologically responsive species. Additionally, this number of animals is consistent with regulatory agency expectations.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Raleigh, NC.
- Age at study initiation: approximately 8 weeks of age.
- Weight at study initiation: the mean: 183 g at receipt and 241 g at randomization; between 226 g and 278 g at the time of dosing.
- Housing: All animals were housed in a study-dedicated, environmentally controlled room, individually in clean, suspended wire-mesh cages. Wire-mesh caging was utilized to acclimate the animals to the wire-mesh flooring utilized in the Nalgene-type metabolism cages utilized during excreta collection. In addition, solid bottom and group housing is not appropriate for radiolabeled studies where the study objectives (quantitative analysis of test article exposure by individual animal) would be compromised by test article exposure from other animals. Cages were elevated above cage-board, which was changed at least 3 times each week. Individual cage cards were affixed to each cage displaying the animal number, group number, and study number. After dosing, animals in the Excretion Mass Balance phases were placed into glass metabolism cages for up to 168 hours. Enrichment devices were provided to each animal for environmental enrichment and to aid in maintaining the animals’ oral health. Devices were not used in the metabolism cages because the debris from the use of the device interferes with the analysis of the feces. The facilities at Charles River are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (block) was offered ad libitum during acclimation and the biological phase of the study.
- Water (e.g. ad libitum): Reverse osmosis-treated water was available ad libitum. Water was provided using an automatic water system. Water bottles were used for animals placed into metabolism cages.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 73°F ± 5°F (22°C ± 3°C). The actual daily mean temperature ranged from to 71.9°F to 72.9°F (22.2°C to 22.7°C).
- Humidity (%): 50% ± 20%. The actual mean daily humidity ranged from 43.0% to 59.5%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: receipt: 07 and 14 of June 2016 To: scheduled euthanasia: 7 days after the dosing

Route of administration:

other: a single oral (gavage) or intravenous (IV, bolus) dose

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to dose preparation, the vial containing [14C]-test article (MET-1015A) was placed under a gentle stream of nitrogen to evaporate the ethanol almost to dryness. Using a syringe, the [14C]-test article was re-suspended with 2 mL of sterile 0.9% saline. The vial was capped, the contents inverted and vortex-mixed to ensure complete dissolution of [14C]-test article.
The solution was assigned reference no. 387082MA1-2-1.
For the Group 1/Group 3 dose formulation, a balance was used to weigh 1.1384 g of test article (TMS No. 150355) into a glass container previously calibrated to contain 38 mL with a stir bar. Using a syringe, 10 mL of sterile 0.9% saline was added to the same glass container. Using a pipette, 0.7 mL of the [14C]-test article stock solution (reference no. 387082MA1-2-1) was added to the same glass container and the contents stirred using a magnetic stir bar and stir plate. While the contents were stirring, the container was filled to the calibration mark using sterile 0.9% saline. The formulation was assigned reference no. 387082MA1-3-1 and continued to mix throughout use. The formulation was stored frozen at approximately -20°C when not in use.
For the Group 2 dose formulation, a balance was used to weigh 0.1033 g of test article (TMS No. 150355) into a glass container previously calibrated to contain 7.5 mL with a stir bar. Using a syringe, 2.5 mL of sterile 0.9% saline was added to the same glass container. Using a pipette, 0.7 mL of the [14C]-test article stock solution (reference no. 387082MA1-2-1) was added to the same glass container and the contents stirred using a magnetic stir bar and stir plate. While the contents were stirring, the container was filled to the calibration mark using sterile 0.9% saline. The formulation was sterile-filtered into a sterile septum-sealable container while in a Laminar flow hood. The formulation was assigned reference no. 387082MA1-4-2 and stored frozen at approximately -20°C when not in use.
For the Group 4/Group 5 dose formulation, a balance was used to weigh 0.9580 g of test article (TMS No. 150355) into a glass container previously calibrated to contain 32 mL with a stir bar. Using a syringe, 10 mL of sterile 0.9% saline was added to the same glass container. Sterile 0.9% saline was used to transfer the remaining 0.6 mL of the [14C]-test article stock solution (reference no. 387082MA1-2-1) into the same glass container and the contents stirred using a magnetic stir bar and stir plate. While the contents were stirring, the container was filled to the calibration mark using sterile 0.9% saline. The formulation was assigned reference no. 387082MA1-7-1 and continued to mix throughout use. The formulation was stored frozen at approximately -20°C when not in use.
The radiochemical concentration and purity of the Group 1/Group 3 [14C]-test article dose formulation (reference no. 387082MA1-3-1) was assessed prior to and following dose administration.

Duration and frequency of treatment / exposure:

single administration.

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Pharmacokinetic phase (in case of iv route of exposure)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

For all phases of the study (in case of oral route of exposure)

No. of animals per sex per dose / concentration:

12/12 for pharmacokinetic phase(oral / iv routes), 2 for the Preliminary Excretion Balance (Pilot) Phase, 4 for the Excretion Mass Balance Phase, and 7 for the Tissue Distribution (QWBA) Phase

Control animals:

not specified

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Animals were assigned to study at random using a computer program. One animal not dosed with test material was used for matrix.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: At approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours postdose, blood samples of approximately 0.5 mL were collected from 4 animals/time point

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, cage washes, plasma and expired air.
- Time and frequency of sampling: Following dosing, Group 3 animals were placed into individual glass metabolism cages equipped for separate collection of expired air, urine, and feces. CO2 trapping solutions were collected at approximately 0 to 6, 6 to 12, 12 to 24, 24 to 36, and 36 to 48 hours postdose. At the end of each CO2 collection interval, the interior surface of the collection tube was rinsed with MeOH and the rinse added to the CO2 trapping solution collection.The activated carbon trap was collected at 24 and 48 hours postdose. Urine was collected on wet ice at approximately 0 to 6, 6 to 12, 12 to 24, and 24 to 48 hours postdose. At the end of each urine collection interval, the interior surface of the cage walls was rinsed with DI water and the rinse retained as a separate sample. Feces were collected on wet ice at approximately 0 to 12, 12 to 24, and 24 to 48 hours postdose (Group 3: Preliminary Excretion Balance (Pilot) Phase).

Following dosing, Group 4 animals were placed into individual glass metabolism cages equipped for separate collection of expired air, urine, and feces. CO2 trapping solutions were collected at approximately 0 to 6, 6 to 12, 12 to 24, 24 to 36, and 36 to 48 hours postdose. At the end of each CO2 collection interval, the interior surface of the collection tube was rinsed with MeOH and the rinse added to the CO2 trapping solution collection. The activated carbon trap was collected at 24 and 48 hours postdose. Urine was collected on wet ice at approximately 0 to 6, 6 to 12, and 12 to 24 hours postdose, and then every approximately 24 hours through 168 hours postdose. At the end of each urine collection, the interior surface of the cage walls was rinsed with DI water and the rinse retained as a separate sample. Feces were collected on ice at approximately 0 to 12 and 12 to 24 hours postdose, and then every approximately 24 hours through 168 hours postdose. After the final urine and feces collection, cages were washed with DI water and the washes retained as a separate sample. Following the final 168 hour collection, animals were euthanized by CO2 inhalation and carcasses weighed and retained in freezer storage (approximately -20°C) for possible analysis (Group 4: Excretion Mass Balance Phase).

At approximately 0.25, 1, 4, 8, 24, 72, and 168 hours postdose, 1 animal/time point was anesthetized with isoflurane and blood samples of approximately 4 mL were collected via cardiac puncture into tubes containing K2EDTA as the anticoagulant. Following blood collection, each anesthetized animal was euthanized by CO2 inhalation and submerged in a dry ice/hexane bath. Frozen carcasses were stored at approximately -20°C until processed for QWBA (Group 5: Tissue Distribution Phase).

- From how many animals: from all animals (samples have been pooled)
- Method type(s) for identification: Liquid scintillation counting (Groups: 1-4); Quantitative Whole Body Autoradiography (QWBA) (Group 5); HPLC-MS (Study Phase: Metabolite Identification)
- Limits of detection and quantification: For each sample analyzed, aliquots of samples collected from undosed animals were fortified with a dilution of the [14C]-test article dosing formulation. Recovery was 95% and 100% for plasma (low and high, respectively) and 97% and 101% for whole blood (low and high, respectively). Excreta recovery ranged from 81% to 112%. The sensitivity of the LSC procedures was estimated using the average background of each sample type assayed. Approximate mean aliquot size and dose were used in the calculations. e quantitation limit in terms of test article equivalents: see table 4 below.
- Other:

Statistics:

Pharmacokinetic Analysis
The individual and/or composite concentration data were used to calculate standard pharmacokinetic parameters such as Area Under the Concentration versus Time Curve (AUClast, AUCinf), peak concentration (Cmax) and the time of its occurrence (Tmax), and elimination half-life (T1/2). Non-compartmental analysis was employed with linear trapezoidal summation for the AUC calculations. The methods and equations for calculation of the pharmacokinetic parameters are presented in the table 5.

Type:

absorption

Results:

Test article is fully absorbed into systemic circulation following oral dosing

Type:

distribution

Results:

Test article is widely distributed predominantly to the adrenal glands, spleen, stomach, pancreas and kidneys.

Type:

metabolism

Results:

The test article was extensively metabolized by the rat following oral administration. Two major metabolites (hydroxylated derivatives) were formed following oral administration. Further metabolites are: N-(OH-butyl)-succinimide and glucuronide conjugate.

Type:

excretion

Results:

The test substance is excreted predominantly via the urine. Approximately 94.1% of the recovered radioactivity was in the urine (including cage rinse), and approximately 4.28% of the dose was eliminated in feces.

Details on absorption:

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in Crl:CD(SD) rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours.
Overall, [14C]-test article-derived radioactivity was widely dispersed, slightly favoring the well-perfused organs and organs associated with oral absorption, and rapidly excreted.

Details on distribution in tissues:

The concentrations of test article equivalents in tissues of male rats are presented in Table 6 (please see attached). [14C]-test article-derived radioactivity was detected in all the tissues examined through 24 hours postdose, except fat, which was quantifiable through 8 hours postdose.
The Cmax for [14C]-test article-derived radioactivity was highest in the adrenal gland (1210 μg-equiv/g) followed by the spleen (632 μg-equiv/g), stomach (484 μg-equiv/g), pancreas (465 μg-equiv/g) and kidney (353 to 374 μg-equiv/g). The remaining tissues were all similar in concentration, ranging from 70 μg-equiv/g (fat) to small intestine (240 μg-equiv/g). There was little notable difference in the concentration of [14C]-test article-derived radioactivity in the tissues. The Tmax was generally 4 hours postdose except for bone and liver (1 hour postdose). Limited data points between Cmax and BLQ precluded calculation of the half-life except in the bone (1.1 h) and liver (5.5 h).
Overall, [14C]-test article-derived radioactivity was widely dispersed, slightly favoring the well-perfused organs and organs associated with oral absorption, and rapidly excreted.
Exposure, as measured by AUClast, generally reflected the order noted for Cmax. Exposure was lowest in fat (<450 h•μg-equiv./g) and highest for the adrenal gland, kidney (all sections), and spleen. Tissue exposure relative to the plasma (tissue:plasma AUClast ratios) were < 1 for most tissues; the highest was 2.26 for the adrenal gland.
Whole blood and plasma data collected from QWBA animals was consistent with the plasma values determined in the PK phase. The whole blood:plasma ratio for AUClast was 0.912, indicating that [14C]-test article-derived radioactivity was evenly distributed between the plasma and blood cells.

Details on excretion:

Based on the excretion data collected during the pilot phase (Group 3), a small fraction (approximately 1%) of the administered dose was captured in the expired air. To ensure mass balance, expired air was also collected in the definitive excretion balance phase (Group 4). Approximately 99.5% of the dose administered to animals in Group 4 was recovered in expired air, urine, and feces from male rats following a single oral administration of [14C]-test article at 300 mg/kg. In agreement with the pilot phase, approximately 1.17% of the recovered radioactivity was in expired air. No volatile components were retained in the activated carbon trap. Approximately 94.1% of the recovered radioactivity was in the urine (including cage rinse), and approximately 4.28% of the dose was eliminated in feces. Because mass balance was achieved, carcasses were not analyzed.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 188 μg-equiv/g at 4 hours postdose

Remarks:

after a single oral dose of [14C]-test article to male SD rats in Group 1 at 300 mg/kg

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: The plasma exposure, AUClast, was 3010 h•μg-equiv./g.

Remarks:

Group 1at 300 mg/kg

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: The terminal elimination phase was well defined (R2 = 0.997); the half-life was 7.96 hours.

Remarks:

Using the terminal elimination phase rate constant, AUCinf was extrapolated and, at 3010 h•μg-equiv./g, was virtually the same as AUClast.

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: After a single IV dose of [14C]-test article to male Crl:CD(SD) rats in Group 2 at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g. The plasma exposure, AUClast, was 212 h•μg-equiv./g.

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: The terminal elimination phase was well defined (R2 = 0.933); the half-life was 9.89 hours. Using the terminal elimination phase rate constant, AUCinf was extrapolated and found to be 213 μg•h/g.

Metabolites identified:

yes

Details on metabolites:

The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose (oral or IV administration) the test article was responsible for < 3% of the radioactivity circulating in plasma. Up to 13 individual metabolites were identified in plasma, plus 1 unknown metabolite and unchanged test article; 8 total metabolites were present at > 5% ROI in at least 1 sample of plasma collected following either route of administration. Two major metabolites were formed. Following oral administration, N-butyl-5-OH-pyrrolidone (M41) increased in relative concentration over the time course and comprised up to 70% ROI in plasma collected at 8 hours postdose (up to 120 μg/g). One other hydroxy metabolite (M35) accounted for up to 11.3% ROI at 4 hours postdose.
Following oral administration, rat urine contained up to 38 metabolites. Ten metabolites were present at > 5% ROI in at least 1 sample. Across all collections, 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (M22, 6%), N-butyl-5-OH-pyrrolidone (M41, 11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (M44, 11%). Parent test article was present at 0.3% of the administered dose in all urine samples combined.
Twelve metabolites were identified in rat feces extractions. Eight metabolites were present at > 5% ROI in at least 1 sample. The summed % of Dose from all of the feces collections analyzed were also calculated; no summed metabolites were present at > 5% of the administered dose. Summed parent test article was present at 0.3% of the dose (please find attached a table with metabolites and their structural formula).

Bioaccessibility (or Bioavailability) testing results:

Based on AUClast (212 h•μg-equiv./g.; Group 2 - iv administration) the absolute oral bioavailability (F) of radioactivity was 142%. Calculated bioavailability > 100% is likely due to an artifact caused by a large difference in the doses administered and does not appear to be based on saturation of metabolic pathways; evaluation of the plasma curves indicates that there is no difference in clearance following administration by either route.

Dose Formulation Analyses and Actual Dose Administered (Table 1, please see attached).

The [14C]-test article dosing formulations administered to rats via oral (gavage) were formulated to provide each animal a target dosage of 300 mg/kg and 200 μCi/kg of radioactivity. The [14C]-test article dosing formulation administered to rats via IV (bolus) injection was formulated to provide each animal a target dosage of 30 mg/kg and 200 μCi/kg of radioactivity. The pre and postdose analyses established homogeneity of the solutions as demonstrated by the low % CV, < 3.6% across triplicate analyses at each interval (Table 3-2, Table 3-3, Table 3-4; Appendix 3). The mean radioconcentration of the formulation for Group 1 and Group 3 on the day of administration was 17.8 μCi/g, or 96.1% of the target. The mean radioconcentration of the formulation for Group 2 on the day of administration was 89.1 μCi/g, or 94.1% of the target. The mean radioconcentration of the formulation for Group 4 and Group 5 on the day of administration was 21.1 μCi/g, or 112.5% of the target.

Radio-HPLC analyses of the dosing solution were performed prior to (all formulations) and after completion of dosing (Group 1 and Group 3, and Group 4 and Group 5 formulations). As indicated by similar pre and postdose radiopurity results, the radiolabeled test article was stable over the dosing period (Table 3-5, Figure 3-1, Figure 3-2, Figure 3-3, Appendix 3). There was no adverse impact resulting from the lack of postdose radiopurity analysis for the Group 2 formulation; the test article stability was adequately established for the other formulations prepared in the same vehicle. The mean radiopurity for each formulation ranged from 98.4% to 98.9%.

The amount of test article to be administered was based on the body weights of the animals on the day of dosing (Table 4-1; Appendix 4). For Group 1, Group 2, and Group 5, the nominal amount delivered was based on the volume administered to each animal. For the purpose of determining mass balance, the actual amount of dosing formulation administered to each animal in Group 3 and Group 4 was quantified by weighing the dosing syringe before and after dosing. Calculations of actual administered doses for individual animals are presented in Table 5-1 (Appendix 5) and mean actual administered dosages of test article are presented in Table 1. Rats received 98% to 102% of the target mass (mg/kg) and 90% to 103% of the target radioactivity (μCi/kg).

Conclusions:

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in Crl:CD(SD) rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours. After a single IV dose of [14C]-test article to male SD rats at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g, AUClast was 212 h•μg-equiv./g, and the half-life was 9.89 hours. The test article is widely distributed to all tissues, and tissue concentrations were correlated to the degree of perfusion by circulating blood. Test article equivalents are rapidly and completely excreted in the urine (94.1%); < 6% of the administered dose was recovered in the feces and expired air.
The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose (oral or IV administration) the test article was responsible for < 3% of the radioactivity circulating in plasma. Two major metabolites were formed: N-butyl-5-OH-pyrrolidone (M41) and 1 other hydroxy metabolite (M35). Rat urine contained up to 38 metabolites; 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (M22, 6%), N-butyl-5-OH-pyrrolidone (M41, 11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (M44, 11%). Parent test article was a minor component of urine, present at 0.3% of the administered dose in all urine samples combined. Twelve metabolites were identified in rat feces extractions and 8 were present at > 5% ROI in at least 1 sample, but no metabolites were present at > 5% of the administered dose. As with urine, parent test article was a minor component (0.3% of the dose).

Executive summary:

The objectives of this study were to determine the plasma pharmacokinetics of [14C]-N-butyl pyrrolidone ([14C]-test article)-derived radioactivity in male Crl:CD(SD) rats following a single oral (gavage) or intravenous (IV, bolus) dose. The routes of elimination and excretion mass balance of [14C]-test article-derived radioactivity, and the tissue distribution and tissue pharmacokinetics of [14C]-test article-derived radioactivity using QWBA methods, were determined in male Crl:CD(SD) rats following a single oral (gavage) dose. Plasma, urine, and fecal homogenate samples were also used for metabolite profiling of [14C]-test article-derived radioactivity.

Study design

For the pharmacokinetic phase, 2 dose groups, each consisting of 12 male rats, received either a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg or a single IV dose of [14C]-test article at 30 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, blood samples were collected from 4 animals/group/time point at approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours. Blood samples were processed to plasma for analysis by LSC. For the preliminary excretion balance (pilot) phase, 1 dose group consisting of 2 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, animals were placed into glass metabolism cages for separate collection of urine, feces, and expired air through 48 hours. For the excretion balance phase, 1 dose group consisting of 4 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, animals were placed into glass metabolism cages for separate collection of expired air through 48 hours and urine and feces through 168 hours. Urine, cage rinse, feces, cage wash, expired air, and carbon trap samples from both excretion balance phases were analyzed by LSC. For the tissue distribution (QWBA) phase, 1 dose group consisting of 7 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. At approximately 0.25, 1, 4, 8, 24, 72, and 168 hours postdose, a whole blood sample was collected from 1 animal/time point. Following blood collection, animals were euthanized by CO2 inhalation and carcasses frozen in a dry ice/hexane bath for analysis by QWBA. Whole blood and plasma samples were analyzed by LSC. Select plasma, urine, and feces samples were selected for radioprofiling and metabolite identification by HPLC-MS/MS with in-line radiodetection.

Results and Key findings

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in male rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours. After a single IV dose of [14C]-test article to male rats at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g, AUClast was 212 h•μg-equiv./g, and the half-life was 9.89 hours. [14C]-test article is widely distributed to all tissues, and tissue concentrations were correlated to the degree of perfusion by circulating blood. At Cmax, most tissue concentrations were within an approximate 5-fold range. Tissue exposure, as measured by AUClast, was lowest in the fat (<450 h•μg-equiv./g) and highest for the adrenal gland, kidney (all sections), and spleen (> 3750 h•μg-equiv./g). Tissue exposure relative to the plasma (tissue:plasma AUClast ratios) were < 1 for most tissues; the highest was 2.26 for the adrenal gland.

Test article equivalents are rapidly and completely excreted in the urine (94.1%); < 6% of the administered dose was recovered in the feces and expired air.

The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose, the test article was responsible for < 3% of the radioactivity circulating in plasma. Two major plasma metabolites were identified: N-butyl-5'-OH-pyrrolidone (M41) and 1 other hydroxy metabolite (M35). Rat urine contained up to 38 metabolites; 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (M22, 6%), N-butyl-5'-OH-pyrrolidone (M41, 11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (M44, 11%). Twelve metabolites were identified in rat feces extractions and 8 were present at > 5% ROI in at least 1 sample, but no metabolites were present at > 5% of the administered dose. Parent test article was a minor component of urine and feces, present at a total of 0.3% of the administered dose in all samples for each matrix.

Description of key information

NBP is expected to be absorbed well after oral exposure, based on the results of the pharmacokinetic study in rats. An extended oral absorption is supported by its low molecular weight, its high water solubility and its optimal Log Pow value. Absorption by inhalation does not play a crucial role. This is based on the low bioavailability for inhalation route due to the low vapour pressure of the substance. However, given its lipophilicity (Log Pow 1.256) - if absorbed - NBP is expected to be absorbed directly across the respiratory tract epithelium or through aqueous pores and/or be metabolized by the alveolar and bronchial tissue. This was confirmed in the inhalation developmental study in rats. Following dermal exposure, NBP is also expected to be absorbed into the stratum corneum and into the epidermis, due to its molecular weight, its optimal Log Pow and high water solubility. The effects observed in the dermal developmental study in rats confirm a certain absorption potential into systemic circulation. The absorption potential through the skin is however significantly lower than the absorption potential via oral or inhalation routes. Concerning distribution in the body, NBP is distributed predominantly to the adrenal glands, spleen, stomach, pancreas and kidneys. The substance does not indicate a significant potential for accumulation. NBP is rapidly metabolised by hydroxylation to hydroxylated products at lactam moiety and N-butylsuccinimide as well as hydroxylated succinimide derivatives. NBP and its metabolites are eliminated mainly via the urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

General

The toxicokinetic profile of N-Butylpyrrolidone (NBP) was studied in an OECD 417 study determining absorption, distribution, metabolism and excretion measurements following a single oral (gavage) or intravenous (IV, bolus) dose of radioactive labelled N-butyl pyrrolidone in male Crl:CD(SD) rats (Charles river, 2016; Report No. 387082). Furthermore, the physico-chemical properties of the substance with the in silico predicted metabolites by the OECD QSAR Toolbox (v.3.3) were integrated with the available toxicological data to create a prediction of the toxicokinetic behavior of NBP. The results of the prediction are completely in line with those observed in the OECD 417 study. Additionally, information on toxicokinetics of two structurally related substances N-Methylpyrrolidone (NMP; CAS 872-50-4) and N-Ethylpyrrolidone (NEP; CAS 2687-91-4) have also been taken into account.

Toxicological profile of NBP

NBP is a colourless liquid (MW 141.2 g/mol) at 20 °C. The substance is completely miscible in water and has a Log Pow of 1.265. Due to the vapour pressure of 13 Pa at 25 °C and the boiling point of 241 °C (at standard pressure of 101.325 kPa), the substance is considered as low volatile compound (according to ECHA guidance R.7c, substances with low volatility have a vapour pressure of less than 0.5 kPa (or a boiling point above 150 °C).

Toxicity study results

An oral LD50 of 300 – 2000 mg/kg bw was established in an acute study with rats (Harlan Laboratories Ltd., 2013; Report No. D41574). Mortalities and clinical signs were observed in treated animals. However, the substance did not produce mortalities, clinical signs or findings at necropsy in an acute dermal study in rats (LD50 >2000 mg/kg bw; Harlan Laboratories, 2014a, Report No. 41401170). NBP is slightly irritating to skin (Charles River Laboratories, 2014a; Report No. 20050801) and irritating to eyes (Charles River Laboratories, 2014b; Report No. 20054210). The effects observed in the three OECD 414 developmental toxicity studies in rats by oral, dermal and inhalation routes of exposure were confined to clinical signs, reduced body weights and food consumption in maternal animals and reduced body weights in fetuses in the highest dose groups. The fertility and developmental parameters were not affected by the test substance administration at all dose levels tested. NOAELs were established at the same dose levels for maternal and developmental toxicity (NOAEC of 600 mg/m³ corresponding to 152.6 mg/kg bw, NOAEL of 500 and 400 for inhalation dermal and oral routes of exposure, respectively). The reproductive toxicity observed at the highest dose levels is considered to represent a secondary non-specific consequence of other toxic effects of the test substance administration.

In the 90-day study (OECD 408), there were treatment related effects observed in animals of either sex in the highest dose group (500 mg/kg bw), and in males of the mid dose group (100 mg/kg bw) (Harlan Laboratories Ltd., 2014b, Report No. 41303953). The effects were adaptive to the treatment but not adverse. No adverse effects were detected during the oestrous cycle assessment and semen parameters assessment. There were no abnormalities detected in reproductive organs, their weights and histopathology. NOAEL of 500 mg/kg bw, the highest dose, was established for both sexes.

Pharmacokinetic study in rats (OECD 417; Charles river, 2016; Report No. 387082)

The plasma pharmacokinetics of [14C]-N-butyl pyrrolidone-derived radioactivity was determined in male Crl:CD(SD) rats following a single oral (gavage) or intravenous (IV, bolus) dose. The routes of elimination and excretion mass balance of [14C]-test article-derived radioactivity, and the tissue distribution and tissue pharmacokinetics of [14C]-test article-derived radioactivity using QWBA methods, were determined in male Crl:CD(SD) rats following a single oral (gavage) dose. Plasma, urine, and fecal homogenate samples were also used for metabolite profiling of [14C]-test article-derived radioactivity.

 

Study design:

For the pharmacokinetic phase, 2 dose groups, each consisting of 12 male rats, received either a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg or a single IV dose of [14C]-test article at 30 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, blood samples were collected from 4 animals/group/time point at approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours. Blood samples were processed to plasma for analysis by LSC. For the preliminary excretion balance (pilot) phase, 1 dose group consisting of 2 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, animals were placed into glass metabolism cages for separate collection of urine, feces, and expired air through 48 hours. For the excretion balance phase, 1 dose group consisting of 4 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. Following dosing, animals were placed into glass metabolism cages for separate collection of expired air through 48 hours and urine and feces through 168 hours. Urine, cage rinse, feces, cage wash, expired air, and carbon trap samples from both excretion balance phases were analyzed by LSC. For the tissue distribution (QWBA) phase, 1 dose group consisting of 7 male rats received a single oral dose of [14C]-test article at 300 mg/kg and a target radioactivity of 200 μCi/kg. At approximately 0.25, 1, 4, 8, 24, 72, and 168 hours postdose, a whole blood sample was collected from 1 animal/time point. Following blood collection, animals were euthanized by CO2 inhalation and carcasses frozen in a dry ice/hexane bath for analysis by QWBA. Whole blood and plasma samples were analyzed by LSC. Select plasma, urine, and feces samples were selected for radioprofiling and metabolite identification by HPLC-MS/MS with in-line radiodetection.

 

Results and Key findings:

Bioavailability and excretion data confirm that [14C]-test article is fully absorbed to systemic circulation following oral dosing in male rats. After a single oral dose of [14C]-test article to male rats in Group 1 at 300 mg/kg, Cmax was 188 μg-equiv/g at 4 hours postdose; AUClast, was 3010 h•μg-equiv./g, and the half-life was 7.96 hours. After a single IV dose of [14C]-test article to male rats at 30 mg/kg, the extrapolated concentration at the time of administration (C0) was 40 μg-equiv/g, AUClast was 212 h•μg-equiv./g, and the half-life was 9.89 hours. [14C]-test article is widely distributed to all tissues, and tissue concentrations were correlated to the degree of perfusion by circulating blood. At Cmax, most tissue concentrations were within an approximate 5-fold range. Tissue exposure, as measured by AUClast, was lowest in the fat (<450 h•μg-equiv./g) and highest for the adrenal gland, kidney (all sections), and spleen (> 3750 h•μg-equiv./g). Tissue exposure relative to the plasma (tissue:plasma AUClast ratios) were < 1 for most tissues; the highest was 2.26 for the adrenal gland.

Test article equivalents are rapidly and completely excreted in the urine (94.1%); < 6% of the administered dose was recovered in the feces and expired air.

The test article was extensively metabolized by the rat following oral administration; by 8 hours postdose, the test article was responsible for < 3% of the radioactivity circulating in plasma. Two major plasma metabolites were identified: N-butyl-5'-OH-pyrrolidone and 1 other hydroxy metabolite associated with OH-N-butylpyrrolidone (IUPAC name: 1-butyl-3-hydroxypyrrolidin-2-one). Rat urine contained up to 38 metabolites; 3 metabolites were present at > 5% of the administered dose: N-(OH-butyl)-succinimide (6%), N-butyl-5'-OH-pyrrolidone (11%), and a glucuronide conjugate of OH-N-butylpyrrolidone (11%). Twelve metabolites were identified in rat feces extractions and 8 were present at > 5% ROI (region of interest) in at least 1 sample, but no metabolites were present at > 5% of the administered dose. Parent test article was a minor component of urine and feces, present at a total of 0.3% of the administered dose in all samples for each matrix.

Toxicokinetic data on structurally similar analogues NMP and NEP

NMP:

The information on the toxicokinetics was obtained from the Annex XV restriction dossier (2013) and is summarized below:

• Studies have been carried out in rats using the dermal, inhalation, oral or intravenous routes.

• 1-methyl-2-pyrrolidone (NMP) is well absorbed following inhalation (40 %-60 %), oral (~100 %) and dermal (≤100 % depending on conditions) exposure (Midgley et al., 1992; Ghantous, 1995; Payan et al., 2002; Kennedy and Delorme, 2004; cited in Annex XV restriction dossier). In humans, NMP is rapidly absorbed following exposure by the inhalation, dermal or oral route in human volunteers (Akesson and Paulsson, 1997; Akesson and Jonsson, 1997; Akesson and Jonsson, 2000; Jonsson and Akesson, 2003; cited in Annex XV restriction dossier, 2013).

• A distribution study following intravenous administration of radiolabelled NMP in the rat showed distribution to all tissues, with highest levels of radioactivity being observed in the liver, bile and small intestine, kidneys, stomach and testis (Wells and Digenis, 1988; cited in Annex XV restriction dossier, 2013). Abstracts of kinetic investigations revealed indications that NMP is able to pass the placenta when pregnant rats were exposed by inhalation or treated orally by gavage. The concentrations found in fetal organs correspond to those of the maternal organs (Sitarek, 2003; Ravn-Jonsen et al., 1992; cited in Annex XV restriction dossier, 2013). About 80 % of the administered dose is excreted as NMP and NMP metabolites within 24h, mainly via the kidneys. The major metabolite is 5-hydroxy-N-methyl-2- pyrrolidone (5-HNMP). Studies in humans show that NMP is rapidly transformed by hydroxylation to 5-HNMP, which is further oxidized to N-methylsuccinimide (MSI); this intermediate is further hydroxylated to 2-hydroxy-N-methylsuccinimide (2-HMSI). The excreted amounts of metabolites in the urine after inhalation or oral intake represented about 100 % and 65 % of the administered doses, respectively.

NEP:

The information on the toxicokinetics was obtained from the registration dossier (CSR) and is summarized below:

NEP (MW 113.16 g/mol) is a colorless liquid, which is miscible with water in any ratio. The Log Pow amounts to -0.2 at 20 °C, indicating that a general accumulation potential of NEP is unlikely. Regarding absorption, NEP is assumed to be bioavailable via all routes as demonstrated by effects in an acute inhalation toxicity study, in a 90-day oral repeated dose toxicity and especially in the oral and dermal teratogenicity studies according to OECD 414. In the acute inhalation study no mortality occurred. Clinical signs like accelerated respiration, squatting posture and a poor general condition on the first day were noted. Body weight gain was reduced. Necropsy revealed nothing unusual. LC50 (aerosol) was > 5.1 mg/L/4h (BASF, 2005, cited in the CSR for NEP). In the 90-day study, substance-related adverse effects were observed in both sexes (BASF, 2006; cited in the CSR on NEP). The target organs were liver and kidneys. In the developmental studies, signs of prenatal developmental toxicity (embryotoxicity and teratogenicity) did occur at dose levels, which were also clearly toxic to the dams (BASF, 2005f, 2007a,b; BASF SE, 2010; Saillenfait et al., 2007; cited in the CSR on NEP)
No dermal absorption study is available. However, comparing the maternal toxicity of the oral and dermal teratogenicity studies in rat and rabbit it can be concluded that absorption via skin takes place to a minor extent than via the oral route. Maternal toxicity (LOAEL) in the rat was 50 mg/kg/day via the oral and 400 mg/kg via the dermal route. In the rabbit, maternal toxicity was 200 mg/kg/ day via the oral and 1000 mg/kg via the dermal route. In a dermal absorption study with the close analogue 1-methyl-2 -pyrrolidone (CAS-No. 872-50-4) 31.6 % of the dose had been absorbed and transported away from the dose site at 24 hours after dosing.

Regarding the main metabolism pathway, NEP is expected to be rapidly biotransformed by hydroxylation to 5-hydroxy-N-ethyl-2-pyrrolidone, which will be further oxidized to N-ethylsuccinimide. This intermediate is subsequently hydroxylated to 2-hydroxy-N-ethylsuccinimide.

All the potential abovementioned metabolites are at least slightly more water soluble than the parent chemical and have a molecular weight lower than 500 g/mol. Therefore, NEP and its metabolites are expected to be excreted predominantly via the urine. Excretion via urine is proven by orange or reddish discolored urine which was observed in all substance-treated rabbits in an oral teratogenicity study. This is most likely due to the excreted test compound or its metabolites.

Toxicokinetic analysis of NBP

Absorption

 

Oral absorption:

NBP was shown to be well absorbed via oral route of exposure in the pharmacokinetic study in rats (OECD 417; Charles river, 2016; Report No. 387082). The prediction based on physico-chemical properties of NBP supports the results of the OECD 417 study. The physico-chemical properties of NBP are in the range suggestive of absorption from the gastrointestinal tract. Oral absorption is favoured for small water-soluble molecules with MW up to 200 g/mol which can pass through aqueous pores with the bulk passage of water (TGD, Part I, Appendix IV, 2003). Therefore, due to the molecular weight of 141.2 g/mol, the high water solubility and the moderate Log Pow value, NBP is expected to be readily absorbed via the gastrointestinal (GI) tract by passive diffusion or by passage through aqueous pores. An extensive oral absorption is also supported by the findings of the acute oral toxicity study, oral 90-day study and an oral developmental study. The effects observed in these studies demonstrate a favoured systemic bioavailability of NBP. Moreover, Human Intestinal Absorption (HIA) of 93.8 % was calculated by the OECD QSAR Toolbox (v3.1). Additionally, the analogue substances NMP and NEP given orally were absorbed rapidly in humans and in animals (see above). Therefore, 100 % oral absorption is considered appropriate for the purposes of hazard assessment (DNEL derivation).

Absorption by inhalation:

Based on the low vapour pressure of NBP (13 Pa at 25 °C), exposure by inhalation is not relevant for this substance. According to ECHA guidance R.7C (Table R.7.12-2), N-Butylpyrrolidone is a substance with low volatility because it has a vapour pressure of less than 0.5 kPa (and boiling point above 150 °C). These parameters indicate that the substance may be not available for inhalation as a vapour. Thus, no considerable amounts of the substance can reach the lung. However, when this occurs, the substance is expected to be absorbed directly across the respiratory tract epithelium or through aqueous pores due to the Log Pow of 1.256 and its high water solubility (> 10,000 mg/L). Indeed, the results of the inhalation developmental toxicity study (OECD 414, Charles river, Report No.: WIL-387081) confirm that absorption by inhalation is extensive if the substance was administered as mixture of vapour and aerosol by whole body to rats. Maternal and developmental toxicity was evidenced at the highest dose level of 1.2 mg/L (corresponding to 315.8 mg/kg bw /day). 0.6 mg/L (corresponds to 152.6 mg/kg bw/day) was considered to be the NOAEL for maternal toxicity and embryo/fetal development. This NOAEL is the lowest NOAEL established in the developmental studies with NBP: in contrast oral NOAEL is 400 mg/kg bw and the dermal NOAEL is 500 mg/kg bw in the same strain of rats. Additionally, the results of an acute inhalation study conducted with NEP indicate also a well systemic absorption. Administered as aerosol, NEP produced bad condition and systemic effects in animals treated with 5.1 mg/L.

NBP is not irritating to the mucous membranes of eyes, nose, and throat; thus no intensification of the absorption can be expected due to the irritating properties of the substance. Based on these data, 100 % absorption is considered for inhalation (worst case).

Dermal absorption:

Dermal absorption of NBP can be deduced from the results of the acute dermal study in rats (Harlan Laboratories, 2014; Report No.: 41401170) and the prenatal developmental study by dermal route of exposure in rats (OECD 414; Charles river, 2016; Report No.: WIL-387079). In the acute study, no mortalities, clinical signs or abnormalities at necropsy were noted. Animals showed expected gains in body weight except for two females which showed no gain in body weight or body weight loss during the first week with expected gain in body weight during the second week. In the prenatal dermal developmental toxicity study, adverse effects were noted in the highest dose group (750 mg/kg bw), which were evidenced by body weight and body weight gain losses with corresponding reduced food consumption and reduced gravid uterine weights in maternal animals and reduced fetal weights. NOAEL of 500 mg/kg bw established for maternal and developmental toxicity is more than twice higher than the NOAEL of 152.6 mg/kg bw (=0.6 mg/L) established in the inhalation prenatal developmental study.

Taking into account only the physico-chemical properties of NBP, the substance is likely to penetrate the skin to a certain extent as the substance is sufficiently lipophilic to cross the stratum corneum (Log Pow of 1.256) and sufficiently soluble in water to partition from the stratum corneum into the epidermis (the substance is fully miscible with water (0 to 100 %)). Absorption through the skin is anticipated to be moderate to high if water solubility lies between 100-10,000 mg/L. However, if water solubility is above 10,000 mg/L (ECHA Guidance R.7c, 2012) and Log Pow value below 0 the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Log Pow of NBP is > 0 (1.265) therefore certain absorption of the chemical through the skin cannot be ruled out. In addition, the molecular weight of 141.2 g/mol indicates a moderate potential to penetrate the skin as well. Additionally, a skin permeability constant Kp of 0.00173 cm/hour was calculated for NBP by EpiSuite (v4.1; dermwin). According to Schumacher et al. (2003), dermal absorption should be regarded as relevant if the permeability constant is <10^–2and ≥10^–4cm/hour. Indeed, the permeability constant of 0.00173 and the results of the dermal prenatal developmental toxicity study indicate that absorption takes place to a certain extent, although no systemic effects were observed in the acute dermal toxicity study. The animals were treated dermally for 24 hours with NBP in the acute study, the time period that is clearly too short to manifest systemic toxicity.

In conclusion, the absorption of NBP via skin is evidenced by the dermal study results and by the physico-chemical properties, but it is less than via the oral and the inhalation routes. Since the irritation potential of NBP is weak and delayed (slight erythema persisted in animals till the end of the observation period) penetration through the damaged skin is not expected to be enhanced to such rates that would result in increased bioavailability. According to TGD, Part I (2003) and ECHA Guidance on Toxicokinetics, Part R.7c (2012), 10 % or 100 % absorption can be assigned for substances in case of absence of substance-specific information on absorption rates through the skin and based only on their physico-chemical properties. However, taking into account lower bioavailability of NBP demonstrated in the acute dermal and prenatal developmental dermal studies, 50 % absorption for dermal route is considered more appropriate than the guidance’s worst-case values. The value of 50 % is, however, is not intended to be used by the DNEL calculations because no route-to-route extrapolation is needed (dermal DNEL for systemic effects will be derived based on NOAEL from dermal developmental toxicity study).

 

Distribution and accumulative potential

Due to the high absorption rate via oral route of exposure, observed in the pharmacokinetic study with NBP a significant amount of NBP is expected to be available for distribution. NBP was widely distributed predominantly to the adrenal glands, spleen, stomach, pancreas and kidneys (OECD 417; Charles river, 2016; Report No.: 387082). The remaining tissues were all similar in concentration. NBP was evenly distributed between the plasma and blood cells. Physico-chemical properties of NBP confirm the ability of the substance to distribute into the cell inner and into the intravascular compartment. As the cell membranes require a substance to be soluble in both water and lipids to be taken up, NBP is expected to reach the inner cell compartment due to its optimal molecular weight of 141.2 g/mol, its Log Pow of 1.256 and a sufficiently high solubility in water (fully miscible (0 to 100 %).

The analogue substances NMP and NEP have a wide distribution throughout the body. In this respect, half-lives of NMP and its metabolites 5-HNMP (hydroxylated at 5th position of lactam ring) and 2-HMSI (2 hydroxy-N-methylsuccinimide) in humans were relatively short: 3.9, 7.5 and 28 hours, respectively (Annex XV restriction dossier for NMP).

Regarding bioaccumulation potential, as it is known that “substances with Log Pow values of 3 or less would be unlikely to accumulate with the repeated intermittent exposure patterns normally encountered in the workplace” (TGD, Part I, 2003), no enhanced risk for accumulation is associated with NBP. Moreover, NBP was rapidly excreted predominantly via the urine in the pharmacokinetic study in rats. Based on these data, no bioaccumulation potential is considered for NBP.

 

Metabolism

NBP was extensively metabolized by the rat following oral administration; by 8 hours postdose (oral or IV administration) in the pharmacokinetic study in rats (OECD 417; Charles river, 2016; Report No.: 387082).

In analogy to NMP and NEP, NBP was oxidized at lactam moiety to the corresponding “hydroxy-pyrrolidone” that was further oxidized to N-butylsuccinimide (1-butylpyrrolidine-2,5-quinone; CAS 3470-96-0). This intermediate was further hydroxylated to hydroxy-N-butylsuccinimide derivatives which was however only aminor metabolite found. The major metabolite N-butyl-5’-OH-pyrrolidone determined in the pharmacokinetic study is fully identical with that predicted by the “Rat liver S9 metabolism simulator” of the OECD QSAR Toolbox (v.3.3). The second hydroxyl metabolite 3-hydroxy-N-butyl-2-pyrrolidone predicted by the OECD QSAR Toolbox was found only at negligible amounts in the urine. The next hydroxylated product identified as di-hydroxy-des-butylpyrrolidone in faeces could correspond to the predicted 3,5-dihydroxy-N-butyl-2-pyrrolidone that in turn can undergo microbial (by the gut flora) transformation to 1-butyl-3-hydroxypyrrolidine-2,5-dione (a hydroxylated derivative of N- butylsuccinimide). The last is a structural analogue of 2-hydroxymethylsuccinimide identified in metabolism studies with NMP.

According to metabolism simulator of the OECD QSAR Toolbox, NBP can also be hydrolysed to 4-(butylamino)butanoic acid under the acidic conditions of stomach. This metabolite was not identified in the pharmacokinetic study with NBP or, if it is formed, was present at amounts outside the detection limit. In such a case, 4-(butylamino)butanoic acid can be regarded as secondary amine that can undergo oxygenation at the C atom by oxygenases (oxidative dealkylation) forming hydroxylated metabolic products like 4-[(4-hydroxybutyl)amino]butanoic acid and 4-[(3-hydroxybutyl)amino]butanoic acid, aldehyde derivative 4-[(4-oxobutyl)amino]butanoic acid or 4,4'-iminodibutanoic acid (dicarboxylic amine acid). Aldehydes originated from dealkylation can be involved into intermediary metabolism (β-oxidation).

 

Excretion

According to the pharmacokinetic study results (OECD 417; Charles river, 2016; Report No.: 387082), approximately 99.5% of the dose administered to animals was recovered in expired air, urine, and feces, whereby 94.1% of the recovered radioactivity was found in the urine, and approximately 4.28% of the dose was eliminated in feces. As NBP and its identified metabolites are sufficiently hydrophilic substances, they are filtered by the kidneys and undergo primarily urinary excretion. Similarly, about 80 % of the administered dose of NMP was excreted as NMP and NMP metabolites mainly via the urine whereby the excreted amounts of metabolites in the urine after inhalation or oral uptake represented about 100 % and 65 % of the administered doses, respectively (Annex XV restriction dossier on NMP, 2013). Metabolites that can re-enter the systemic circulation are not expected to occur according to the pharmacokinetic study results.