4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jun 2016 to 05 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Objective of study:

absorption

distribution

excretion

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC 1107/2009

Version / remarks:

October 2009

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EC 283/2013

Version / remarks:

March 2013

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 Nohan No 8147

Version / remarks:

Nov 2000

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Cas Number:

2061933-85-3

Molecular formula:

C23H19Cl2F4N3O4

IUPAC Name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid: particulate/powder

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Remarks:

(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Groups 1 - 6: 8 - 9 weeks and Groups 7 and 8: 10 - 11 weeks
- Weight at study initiation: 228 - 252 g and 156 - 182 g for males and females, respectively (Group 1)
267 - 290 g and 176 - 196 g for males and females, respectively (Group 2); 238 g and 159 g for male and female, respectively (Group 3); 275 g and 166 g for male and female, respectively (Group 4); 237 g and 152 g for male and female, respectively (Group 5); 229 g and 149 g for male and female, respectively(Group 6); 283 - 327 g and 199 - 211 g for males and females, respectively (Group 7); 268 - 290 g and 194 - 230 g for males and females, respectively (Group 8).
- Housing: Pre-study: Multiply housed by sex in solid bottomed polycarbonate cages with bedding. On study: Singly in all-glass metabolism cages
- Diet: A standard laboratory diet of known formulation available ad libitum.
- Water: Mains tap water ad libitum
- Acclimation period: At least 5 days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 38 - 67
- Air changes per hr: minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 Jun 2016 To: 05 Jan 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % (w/v) with/without 0.5% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to dose preparation, the radiochemical purity of the stock [14C]-test substance was determined by HPLC and TLC methods.

TRIAL ORAL PREPARATIONS
Prior to dosing, trial [methylphenyl-14C], [halophenyl-14C] and [oxoisoxazolidinyl-14C]-test substance oral formulations were prepared at target dose concentrations of 0.2 and 2 mg/mL. The suitability of the dose formulation procedures and radiochemical stability of [methylphenyl-14C]-test substance was assessed in the dose preparations at 1, 8 and 15 days post preparation and [halophenyl-14C] and [oxoisoxazolidinyl-14C]-test substance assessed at 24 and 48 hours post preparation by HPLC and TLC. Due to technical issues the 0.2 mg/mL [oxoisoxazolidinyl-14C]-test substance preparation was assessed by HPLC at 53 hours rather than 48 hours post preparation. The isomeric ratio was measured by HPLC.. The trial dose preparations mimicked the procedures required for the main animal study, but were prepared using the minimal practical quantities of test substance.
The trial doses were stored in a fridge set to maintain 4 °C when not in use. All dose preparations were stored in a fridge set to maintain 4 °C when not in use. For the 0.2 mg/mL preparations, an accurate volume of [14C]-test substance stock solution was transferred into a mortar bowl.
For the 2 mg/mL preparations, an appropriate amount of unlabelled test substance was accurately weighed into a volumetric flask. An appropriate volume of [14C]-test substance stock solution was accurately dispensed into the volumetric flask containing the unlabelled test substance. The flask was made up to volume using acetonitrile and aliquots taken for specific activity confirmation. The contents of the flask, along with washings, were transferred directly into a mortar bowl.
The contents of each mortar bowl were evaporated to dryness under a steady stream of nitrogen. The remaining [14C]-test substance was lightly wetted with an appropriate amount of dose vehicle (0.5 % aqueous CMC for Groups 5 & 6, 0.5 % aqueous CMC containing 0.5 % Tween 80 for Groups 1 - 4, 7 & 8) and ground into a fine paste with a pestle. Additional dose vehicle was added and the paste was transferred to a pre-weighed glass container. This process was repeated several times before the contents of the container were made up to the final dose volume to achieve the required target concentration. Aliquots were taken to check the concentration and homogeneity prior to dosing. Whenever practicable, the dose preparation was stirred with a magnetic stirrer until dose preparation and dosing were complete.

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1 - orally dosed with [methylphenyl-14C]

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2 - orally dosed with [methylphenyl-14C]

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3 - orally dosed with [halophenyl-14C]

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4 - orally dosed with [halophenyl-14C]

Doses / concentrations 5

Dose / conc.:

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

Remarks:

Group 5 - orally dosed with [oxoisoxazolidinyl-14C]

Doses / concentrations 6

Dose / conc.:

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

Remarks:

Group 6 - orally dosed with [oxoisoxazolidinyl-14C]

Doses / concentrations 7

Dose / conc.:

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

Remarks:

Group 7 - orally dosed with [methylphenyl-14C]

Doses / concentrations 8

Dose / conc.:

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

Remarks:

Group 8 - orally dosed with [methylphenyl-14C]

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on study design:

A group of 4 male and 4 female rats per dose (methylphenyl label) or 1 male and 1 female rat per dose (halophenyl and oxoisoxazolidinyl labels) were each given either a single oral administration of nominally 1 mg/kg (Groups 1, 3, 5 and 7) or 10 mg/kg (Groups 2, 4, 6 and 8) of [methylphenyl-14C], [halophenyl-14C] and [oxoisoxazolidinyl-14C]-test substance. In each group, excreta samples were taken over predetermined time intervals up to 7 days post dose (Groups 1 - 6) or 3 days post dose (Groups 7 and 8).

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY
Dose administration
Each animal was accurately weighed prior to dosing. The syringes were weighed prior to and following each dosing occasion. The actual dose received by each animal was determined with reference to the radioactive concentration, the weight of dose administered and the calculated or supplied specific activity of the test substance. The oral dose preparations were administered by metal gastric gavage at 5 mL/kg, to achieve target doses of 1 mg/kg (Groups 1, 3, 5 and 7) and 10 mg/kg (Groups 2, 4, 6 and 8). Animals received a target radioactive dose of 5 MBq/kg".

Excretion:
Following oral dosing of intact animals, urine and faeces were frozen upon excretion by collection over solid carbon dioxide (up to 168 hours post dose). Urine was collected predose and at 8 hrs post dose, faeces was collected at predose, and urine and faeces were then collected at daily intervals until termination (168 hrs post dose). The cages were rinsed with ca 5 mL of water prior to the removal of the urine pot. At each faeces collection timepoint post dose, the cages were rinsed with a suitable volume of water and the washes collected separately.
Following dosing of BDC animals, urine, faeces and bile were frozen upon excretion by collection over solid carbon dioxide (up to 72 hours post dose). Bile was collected at the following time points:
Predose, 0 - 1, 1 - 2, 2 - 4, 4 - 8, 8 - 12, 12 - 24, 24 - 48 and 48 - 72 hours post dose.
Urine was collected predose and at 8 hours post dose, faeces was collected at predose, and urine and faeces were then collected at daily intervals until termination (72 hours post dose). The cages were rinsed with ca 5 mL of water prior to the removal of the urine pot. At each faeces collection timepoint post dose the cages were rinsed with a suitable volume of water and the washes collected separately.
The levels of total radioactivity were determined in each sample collected.

Pharmacokinetics:
A terminal blood sample (Groups 1 - 8) (ca 3 - 10 mL) was taken into heparinised tubes and ca 0.5 mL retained for radioanalysis. The remaining blood sample was centrifuged to obtain plasma. In Groups 1 - 8, the gastrointestinal tract (and contents) and residual carcasses were retained separately. The following tissues were also removed from the intact animals (Groups 1 and 2): adrenals, brain, heart, kidneys, liver, lungs, ovaries (females), pancreas, spleen, testes (males), thymus, thyroid and uterus (females) together with representative samples of bone mineral (tibia, fibula), fat (renal) and muscle.
Samples not analysed immediately were stored in a freezer set to maintain -20 °C until taken for analysis with the exception of cage wash, which was stored at ambient temperature and the remaining carcass, which was stored at ambient temperature once digestion solution was added. Following analysis, samples (excluding cage wash and carcass) were returned to storage in a freezer set to maintain -20 °C.
Total radioactivity was determined in each sample collected.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Absorption of [methylphenyl-14C]-test substance was 66 - 68 % following a 1 mg/kg dose and 52 - 53 % following a dose of 10 mg/kg.

Main ADME results 2

Type:

distribution

Results:

Liver and kidney contained the highest level of radiolabelled residues seven days after admission, which is consistent with the urinary and biliary elimination of absorbed [methylphenyl-14C]-test substance.

Main ADME results 3

Type:

excretion

Results:

The majority of the absorbed dose was excreted in faeces via biliary elimination, but excretion was incomplete at 168 h.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

In bile duct cannulated rats (Group 7 and 8), as the [methylphenyl-14C]-test substance dose increased from 1 to 10 mg/kg, absorption was reduced indicating absorption was dose dependent. At each dose level, the absorption was similar in males and females, with oral absorption ranging from 66 - 68 % (1 mg/kg) to 52 - 53 % (10 mg/kg).

Details on distribution in tissues:

Seven days following administration of [methylphenyl-14C]-test substance at 1 or 10 mg/kg to male and female rats, concentrations of radioactivity in all tissues were above that of circulating blood. The highest tissue concentrations were observed in the kidneys with means of 0.589 and 0.527 µg equiv/g in males and females, respectively, following a dose of 1 mg/kg and means of 4.41 and 4.55 µg equiv/g in males and females, respectively, following a dose of 10 mg/kg.

Details on excretion:

Following a single oral dose of [methylphenyl-14C], [halophenyl-14C] or [oxoisoxazolidinyl-14C]-test substance at 1 or 10 mg/kg, the major route of elimination was via the faeces, with urinary elimination fairly minor. Urinary elimination was slightly more prominent in the oxoisoxazolidinyl label. The majority of the administered radioactivity was excreted by 72 h post dose. Excretion was incomplete by 168 h post dose, as indicated by the remaining dose in the terminal samples. A satisfactory quantitative recovery was obtained for all groups.
In bile duct cannulated rats (Group 7 and 8), a single oral dose of [methylphenyl-14C]-test substance at 1 mg/kg revealed the bile as major route, followed by the faeces. At 10 mg/kg of the same test substance, radioactivity was approximately evenly distributed in the faeces and bile. Urinary elimination was minor following both doses.
Excretion was incomplete in both sexes by 72 h post dose with 11-19% remaining in carcass and gastrointestinal tract.

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

ANALYSIS OF DOSE PREPARATION

Prior to dose preparation, the radiochemicals were shown to have a purity of > 96 %. The pre- and post-dose radiochemical purities of [methylphenyl-14C], [halophenyl-14C] and [oxoisoxazolidinyl-14C]-test substance were > 95 % indicating that the test substances were stable during dose preparation and for the duration of the dosing procedure. Analyses of individual aliquots of the dose preparation taken over the course of the study were within 0.4 - 6.7 % of the mean, indicating that a satisfactory homogeneity had been achieved. The group mean achieved oral doses for [methylphenyl-14C]-test substance were 1.07 and 1.10 mg/kg and 9.72 and 10.1 mg/kg for intact males and females, respectively and 1.08 and 1.10 mg/kg and 9.81 and 10.4 mg/kg for cannulated males and females, respectively. The achieved oral doses for [halophenyl-14C]-test substance were 0.978 and 0.964 mg/kg and 9.92 and 9.72 mg/kg for the male and female, respectively. The achieved oral doses for [oxoisoxazolidinyl-14C]-test substance were 0.880 and 0.947 mg/kg and 8.28 and 8.22 mg/kg for the male and female, respectively.

ANIMAL OBSERVATIONS

In the 10 mg/kg BDC Group 8, clinical signs were observed on days 1, 3 & 4 following dosing in one female animal. Signs noted included piloerection, prominent spine and decreased activity. The remaining 3 females also displayed pilo-erection at ca 7 hours post dose on day 1. No adverse observations were noted in any other group.

Table 2: Summary of mean [methylphenyl-14C]-test substance (Groups 1 - 6)

[methylphenyl-14C]-test substance	Group 1 (1 mg/kg)		Group 2 (10 mg/kg)
	Male (n = 4)	Female (n = 4)	Male (n = 4)	Female (n = 4)
	% dose
Total urine (0-168 h)	3.8	3.0	4.7	3.9
Total faeces (0-168 h)	87	86	91	91
Total cage wash (0-168 h)	1.3	1.4	1.3	0.8
0-72 h excretion	81	79	85	79
Total terminal samples (168 h)	7.2	8.4	6.2	7.7
Total recovery (168 h)	99	98	103	104
	Concentration (µg equiv/g or mL)
Whole blood (168 h)	0.037	0.035	0.29	0.31
Plasma (168 h)	0.015	0.016	0.13	0.13

Table 3: Summary of mean [halophenyl-14C]-test substance results (Groups 1 - 6)

[halophenyl-14C] -test substance	Group 3 (1 mg/kg)		Group 4 (10 mg/kg)
	Male (n = 1)	Female (n = 1)	Male (n = 1)	Female (n = 1)
	% dose
Total urine (0-168 h)	2.5	2.2	1.8	3.6
Total faeces (0-168 h)	90	77	98	93
Total cage wash (0-168 h)	0.5	1.3	1.3	0.9
0-72 h excretion	82	69	85	83
Total terminal samples (168 h)	10	12	9.3	9.1
Total recovery (168 h)	104	92	110	107
	Concentration (µg equiv/g or mL)
Whole blood (168 h)	0.025	0.029	0.33	0.25
Plasma (168 h)	0.014	0.013	0.14	0.12

 

Table 4: Summary of mean [oxoisoxazolidinyl-14C]-test substance results (Group 1 - 6):

[oxoisoxazolidinyl-14C]- test substance	Group 5 (1 mg/kg)		Group 6 (10 mg/kg)
	Male (n = 1)	Female (n = 1)	Male (n = 1)	Female (n = 1)
	% dose
Total urine (0-168 h)	8.1	8.4	5.9	5.7
Total faeces (0-168 h)	88	82	84	85
Total cage wash (0-168 h)	1.1	1.6	4.3	3.3
0-72 h excretion	90	85	84	83
Total terminal samples (168 h)	5.5	5.6	5.7	5.8
Total recovery (168 h)	102	98	100	100
	Concentration (µg equiv/g or mL)
Whole blood (168 h)	0.027	0.027	0.29	0.24
Plasma (168 h)	0.009	0.008	0.08	0.08

Table 5: Summary of mean [methylphenyl-14C]-test substance results (Groups 7 - 8)

[methylphenyl-14C]-test substance	Group 7 (1 mg/kg)		Group 8 (10 mg/kg)
	Male (n = 4)	Female (n = 4)	Male (n = 4)	Female (n = 4)
	% dose
Total urine (0-72 h)	1.7	2.9	2.3	3.9
Total faeces (0-72 h)	30	32	47	46
Total bile (0-72 h)	50	46	41	33
Total cage wash (0-72 h)	0.5	0.6	0.5	0.5
% Absorption	68	66	53	52
Total terminal samples (72 h)	17	19	11	18
Total recovery (72 h)	99	100	102	102
	Concentration (µg equiv/g or mL)
Whole blood (72 h)	0.081	0.141	0.48	1.21
Plasma (72 h)	0.073	0.191	0.46	1.73

 

Applicant's summary and conclusion

Conclusions:

Irrespective of dose, radiolabel or sex, following a single oral administration of [14C]-test substance, the majority of dose related radioactivity was eliminated by 72 h post dose, but excretion was incomplete at 168 h. Absorption of [methylphenyl-14C]-test substance was 66- 68% following a 1 mg/kg dose and 52 - 53 % following a dose of 10 mg/kg. The majority of the absorbed dose was excreted in faeces via biliary elimination.
Seven days after administration of [methylphenyl-14C]-test substance, radioactive residues in all tissues were detectable and above that of circulating blood. Higher concentrations in the liver and kidney are consistent with the urinary and biliary elimination of absorbed [methylphenyl-14C]-test substance.

Executive summary:

The absorption, excretion, biliary elimination and tissue distribution of [methylphenyl-14C]-test substance was investigated following oral doses of 1 and 10 mg/kg, to groups of 4 male and 4 female rats. The excretion of [halophenyl-14C] and [oxoisoxazolidinyl-14C]-test substance was also investigated following oral doses of 1 and 10 mg/kg, to 1 male and 1 female rat per radiolabel and dose. Excretion samples were obtained over a 7 day period for intact animals or 3 days for bile duct cannulated animals. After this period, the rats were humanely killed and residual radioactivity was measured in selected tissues (intact only) and remaining carcass. The nature and identity of metabolites present in the bile and excreta were also investigated and reported separately.

Following a single oral dose of [methylphenyl-14C], [halophenyl-14C] or [oxoisoxazolidinyl-14C]-test substance to intact rats at 1 or 10 mg/kg, the major route of elimination was via the faeces, with urinary elimination fairly minor. The routes and rates were similar regardless of sex, dose or radiolabel position. Urinary elimination was slightly more prominent in the oxoisoxazolidinyl label. The majority of the administered radioactivity was excreted by 72 h post dose. Excretion was incomplete by 168 h post dose, as indicated by the remaining dose in the terminal samples. A satisfactory quantitative recovery was obtained for all groups. Seven days after administration of 1 or 10 mg/kg [methylphenyl-14C]-test substance, radioactive residues in all tissues were detectable and above that of circulating blood. The highest mean tissue concentration was observed in the kidneys, with the tissue distribution of radioactivity being similar in both sexes following both doses. Higher concentrations in the liver and kidney are consistent with the urinary and biliary elimination of absorbed [methylphenyl-14C]-test substance.

Following a single oral dose of [methylphenyl-14C]-test substance to bile duct cannulated rats at 1 mg/kg, the major route of elimination was via the bile, followed by the faeces. Following a single oral dose of [methylphenyl-14C]-test substance to bile duct cannulated rats at 10 mg/kg, radioactivity was approximately evenly distributed in the faeces and bile. Urinary elimination was minor following both doses. The routes and rates were broadly similar regardless of sex at each dose. The mean oral absorption was reduced following an increase in dose from 1 to 10 mg/kg, indicating absorption was dose dependent. At each dose level, the absorption was similar in males and females. Excretion was incomplete in both sexes by 72 hr post dose. The total mean recovery of administered radioactivity was quantitative.

Irrespective of dose, radiolabel or sex, following a single oral administration of [14C]-test substance, the majority of dose related radioactivity was eliminated by 72 hr post-dose, but excretion was incomplete at 168 h. Absorption of [methylphenyl-14C]-test substance was 66 - 68 % following a 1 mg/kg dose and 52-53% following a dose of 10 mg/kg. The majority of the absorbed dose was excreted in faeces via biliary elimination. Seven days after administration of [methylphenyl-14C]-test substance, radioactive residues in all tissues were detectable and above that of circulating blood. Higher concentrations in the liver and kidney are consistent with the urinary and biliary elimination of absorbed [methylphenyl-14C]-test substance.