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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

July 2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

other: EC 1107/2009

Version / remarks:

October 2009

Qualifier:

according to guideline

Guideline:

other: EC 283/2013

Version / remarks:

March 2013

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 Nohan No 8147

Version / remarks:

Nov 2000

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

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

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

Dose / conc.:

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

Remarks:

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

Dose / conc.:

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

Remarks:

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

Dose / conc.:

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

Remarks:

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

Dose / conc.:

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

Remarks:

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

Dose / conc.:

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

Remarks:

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

Dose / conc.:

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

Remarks:

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

Dose / conc.:

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

Remarks:

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

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.

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.

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.

Type:

excretion

Results:

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

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.

Metabolites identified:

no

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

 

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.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Aug 2016 to 13 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

other: pharmacokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

July 2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

other: EC No 1107/2009

Version / remarks:

21 October 2009

Qualifier:

according to guideline

Guideline:

other: EU No. 283/2013

Version / remarks:

01 March 2013

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147

Version / remarks:

November 24, 2000

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

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 - 3: 8 - 9 weeks
- Weight at study initiation: 232 - 276 g and 161 - 197 g for males and females, respectively (Group 1); 253 - 304 g and 160 - 197 g for males and females, respectively (Group 2); 259 - 296 g and 164-196 g for males and females (Group 3).
- Housing: pre-study: multiply housed by sex in solid bottomed polycarbonate cages with wood shavings. On study: multiply housed by sex in polycarbonate and stainless steel cages with raised wire mesh floors.
- Diet: a standard laboratory diet of known formulation was available ad libitum.
- Water: mains tap water was supplied ad libitum.
- Acclimatisation: At least 5 days.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 21 - 22
- Humidity (%): 43 - 62
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Aug 2016 To:13 Sep 2016

Route of administration:

other: oral and intravenous

Vehicle:

other: Oral: 0.5% (w/v) aqueous CMC containing 0.5% Tween 80. For invravenous: DMSO: PEG 400: 20% aqueous hydroxypropyl-β-cyclodextrin (10:20:70 (v/v/v)).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Prior to dose preparation, the radiochemical purity of the stock [14C]-labeled test substance was determined by HPLC and TLC methods.
- Samples of blood were prepared for LSC by sample oxidation as follows: duplicate samples were oxidised in a Sample Oxidiser. The [14C]-carbon dioxide generated was absorbed in Carbosorb (8 mL) to which scintillation fluid (10 mL) was added and then analysed by liquid scintillation counting. Combustion of standards showed that recovery efficiencies were close to, or were in excess of 97 % throughout.
- The homogeneity of the dose preparation and the level of radioactivity in dose residues and plasma samples were determined by Liquid Scintillation Counting (LSC) as follows: all samples prepared in scintillant were analysed for 5 minutes, together with representative blank and standard vials, using a liquid scintillation analyser with automatic quench correction using an external standard method. Samples were analysed in duplicate and allowed to heat and light stabilise prior to analysis. Disintegrations per minute (dpm) values were calculated using the appropriate quench correction data.

ANALYSIS OF THE DOSE PREPARATIONS
- Radiochemical purity was determined by both HPLC and TLC methods. The radiochemical purity was determined in the dose preparations prior to and after dosing. To measure the radioactive concentration and homogeneity in the dose preparations, six weighed aliquots (3 prior to dosing and 3 after dosing) from each preparation were dispensed into 25 mL volumetric flasks and dissolved in acetonitrile or ethanol. Duplicate aliquots of each dilution were analysed by LSC.

DOSE PREPARATION
- Prior to dose preparation, the radiochemical purity of the stock [14C]-test substance was determined by HPLC and TLC methods. The oral preparations were previously shown to be stable for up to 15 days in another study.
- Trial intravenous preparation: prior to dosing, a trial [14C]-test substance intravenous formulation was prepared at a target dose concentration of 0.2 mg/mL to assess the suitability of the dose formulation procedures and radiochemical stability of [14C]-test substance in the dose preparation at 3 and 24 hours post preparation by HPLC and TLC as detailed in Appendix 4. The isomeric ratio was measured by HPLC. The trial dose preparation mimicked the procedures required for the main animal study, but were prepared using the minimal practical quantities of test substance.
- Oral dose preparations: for the Group 1 preparation, an accurate volume of [14C]-test substance stock solution was transferred into a mortar bowl. For the Group 2 preparation, 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 containing 0.5% Tween 80) and ground into a fine paste with a pestle. Additional dose vehicle was added and the paste was transferred to a preweighed 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.
- Intravenous dose preparations: for the intravenous dose preparation, an appropriate volume of [14C]-test substance stock solution was accurately dispensed into a pre-weighed glass container. The contents were evaporated to dryness under a steady stream of nitrogen. Appropriate volumes of DMSO and PEG 400 were added and shaken until mixed. An appropriate volume of 20 % aqueous HPβCD was added gradually whilst stirring to a final ratio of 10:20:70 respectively. Aliquots were taken to check the concentration and homogeneity prior to dosing

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

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

Remarks:

Group 1: oral dose (gavage)

Dose / conc.:

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

Remarks:

Group 2: oral dose (gavage)

Dose / conc.:

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

Remarks:

Group 3: intravenous dose

No. of animals per sex per dose / concentration:

Oral doses: 20
Intravenous: 4

Control animals:

no

Details on study design:

Groups of 20 male and 20 female rats were each given either a single oral dose of [14C]-test substance at nominally 1 mg/kg (Group 1) or 10 mg/kg (Group 2). A group of 4 male and 4 female rats (Group 3) were given a single intravenous dose at nominally 1 mg/kg of [14C]-test substance. Serial or terminal blood samples were taken over predetermined time intervals up to 4 days post dose in each oral group and up to 3 days post dose in each iv group.

Details on dosing and sampling:

Blood samples were collected from each rat (into blood tubes containing lithium heparin anticoagulant) at defined intervals following dosing. For more information see 'Any other information on materials and methods incl. tables'.

Preliminary studies:

ANALYSIS OF DOSE PREPARATION
Prior to dose preparation the radiochemical was shown to have a purity of > 96 % as determined by HPLC and TLC. Prior to intravenous dose preparation the radiochemical stability of [14C]-test substance was assessed at 3 and 24 h post preparation by HPLC and TLC and was found to be > 96 %. The pre- and post-dose radiochemical purities of [14C]-test substance were > 96 %, as determined by HPLC and TLC, indicating that the test item was stable during dose preparation and for the duration of the dosing procedure. The isomeric ratio in the intravenous trial was determined by HPLC as 92.5: 1.5: 5.8: 0.1 (isomer A: isomer B: isomer C: isomer D).

Analyses of individual aliquots of the dose preparations taken over the course of the study were within 3.1 - 5.5 % of the mean, indicating that a satisfactory homogeneity had been achieved. The group mean achieved doses for [14C]-test substance were 1.06 and 1.14 mg/kg (Group 1) and 9.96 and 10.0 mg/kg (Group 2) for oral group males and females, respectively, and 1.02 and 1.04 mg/kg (Group 3) for intravenous group males and females, respectively.

Type:

absorption

Results:

The absorption of total radioactivity was > 88 % following oral administration of [14C]-test substance, irrespective of dose and sex

Details on absorption:

1 MG/KG ORAL DOSE (GROUP 1)
Following a single oral dose of 1 mg/kg [14C]-test substance, the peak concentration (Cmax) of radioactivity was observed in blood at 8 hours post dose in males (0.411 µg equiv/g) and 6 hours in females (0.418 µg equiv/g). Thereafter, concentrations in blood declined to 0.046 µg equiv/g in males and 0.058 µg equiv/g in females at 96 hours. The AUC(0-t) was 15.3 and 16.6 µg equiv.h/g, in males and females, respectively. The apparent terminal phase half life (t1/2) was 34 hours in males and 39 hours in females, with the AUC(0-inf) 17.6 and 19.9 µg equiv.h/g in males and females, respectively (13 - 16 % extrapolated).
The Cmax of radioactivity in plasma was observed at 8 hours post dose in males (0.723 µg equiv/mL) and 6 hours in females (0.664 µg equiv/mL). Thereafter, concentrations in plasma declined to 0.039 µg equiv/mL in males and 0.046 µg equiv/mL in females at 96 hours. The AUC(0-t) was 22.2 and 23.6 µg equiv.h/mL, in males and females, respectively. The apparent terminal phase half life (t1/2) was 25 hours in males and 26 hours in females, with the AUC(0-inf) 23.6 and 25.4 µg equiv.h/mL in males and females, respectively (5.9 - 6.9 % extrapolated). Blood to plasma ratios were in the range 0.5-1.2 in males and 0.5 - 1.3 in females. The blood to plasma ratios of radioactivity indicated that the radioactivity was more associated with the plasma fraction up to 48 hours post dose in males and females then became increasingly associated with the cellular fraction to 96 hours. The oral bioavailability (F) was 96% in males and 100 % in females.

10 MG/KG ORAL DOSE (GROUP 2)
Following a single oral dose of 10 mg/kg [14C]-test substance to male and female rats, the peak concentration (Cmax) of radioactivity was observed in blood at 12 hours post dose in males (2.71 µg equiv/g) and 4 hours in females (2.60 µg equiv/g). Thereafter, concentrations in blood declined to 0.55 µg equiv/g in males and 0.57 µg equiv/g in females at 96 hours. The AUC(0-t) was 132 and 128 µg equiv.h/g, in males and females, respectively. In males, the apparent terminal phase half life (t1/2) was 39 hours, with the AUC(0-inf) in males of 164 µg equiv.h/g (19% extrapolated). However, in females an apparent terminal phase could not be reliably estimated.
The Cmax of radioactivity in plasma was observed at 12 hours post dose in males (5.01 µg equiv/mL) and 4 hours in females (4.18 µg equiv/mL). Plasma concentrations of radioactivity then steadily declined to 0.55 µg equiv/mL in both males and females at 96 hours. The AUC(0-t) was 198 and 179 µg equiv.h/mL, in males and females, respectively. The apparent terminal phase half life (t1/2) was 30 hours in males and 29 hours in females, with the AUC(0-inf) 222 and 202 µg equiv.h/mL in males and females, respectively (11 % extrapolated). Blood to plasma ratios were in the range 0.5 - 1.0 in males and 0.6 - 1.1 in females. The blood to plasma ratios of radioactivity indicated that the radioactivity was more associated with the plasma fraction up to 72 hours post dose in both males and females, and was approximately evenly distributed at 96 hours. The oral bioavailability (F) was 88 % in both males and females.

1 MG/KG INTRAVENOUS DOSE (GROUP 3)
Following a single intravenous dose of 1 mg/kg [14C]-test substance to male and female rats, the theoretical concentration of radioactivity in blood at time zero (C0) was 0.306 and 0.308 µg equiv/g, in males and females, respectively. Thereafter, blood concentrations plateaued to 24 hours post dose, then steadily declined to 72 hours post dose (0.107 µg equiv/g for males and 0.103 µg equiv/g for females). The AUC(0-t) was 15.4 µg equiv.h/g in males and 15.2 µg equiv.h/g in females. In males an apparent terminal phase could not be reliably estimated. In females, the apparent terminal phase half life (t1/2) was 26 hours, with the AUC(0-inf) 16.7 µg equiv.h/g (18 % extrapolated). Other PK parameter estimates (MRT, Vss, CL) following iv administration can be found in Table 5 in 'Any other information on results incl. tables'.

Metabolites identified:

no

Animal observations

No adverse animal observations were noted in this study.

Table 3: Mean Blood Concentrations and Pharmacokinetic Parameters Following Single Oral Administration of [14C]-test substance to Rats Expressed as µg Equivalents of test substance /g

Nominal Time after dosing (h)	Group 1 – 1 mg/kg oral		Group 2 – 10 mg/kg oral
	Male	Female	Male	Female
0.5	0.103	0.121	0.54	0.88
2	0.282	0.285	1.95	2.03
4	0.370	0.344	2.60	2.60
6	0.356	0.418	2.45	1.95
8	0.411	0.351	2.54	1.90
12	0.351	0.335	2.71	2.26
24	0.221	0.256	1.77	1.74
48	0.122	0.137	1.29	1.34
72	0.074	0.088	0.81	0.86
96	0.046	0.058	0.55	0.57
Cmax(µg equiv/g)	0.411	0.418	2.71	2.60
Cmax/D	0.388	0.366	0.272	0.260
tmax (hours)	8	6	12	4
t1/2 (hours)	34.4	38.8	39.2	39.1^*
AUC(0-t) (µg equiv.h/g)	15.3	16.6	132	128
AUC(0-t)/D	14.5	14.6	13.3	12.8
AUC(0-inf) (µg equiv.h/g)	17.6	19.9	164	161^*
AUC(0-inf)/D	16.6	17.4	16.4	16.1^*
AUC % Extrap	13.0	16.4	19.1	20.1^*
F (%)	95.6	99.6	87.8	87.6

PK parameters are Phoenix (WinNonlin) derived.

Note: Dose normalised parameters were calculated using mean actual doses.

Note: F was calculated using AUC_(0-t)for Groups 1 and 2.

* = The extrapolation of the AUC to infinity represents more than 20 % of the total area

Table 4: Mean Plasma Concentrations and Pharmacokinetic Parameters Following Single Oral Administration of [14C]-test substance to Rats Expressed as µg Equivalents of test substance/mL

Nominal Time after dosing (h)	Group 1 – 1 mg/kg oral		Group 2 – 10 mg/kg oral
	Male	Female	Male	Female
0.5	0.197	0.225	0.88	1.50
2	0.553	0.585	3.48	3.46
4	0.637	0.540	4.62	4.18
6	0.578	0.664	4.64	3.10
8	0.723	0.624	4.51	3.14
12	0.581	0.643	5.01	3.85
24	0.331	0.361	2.78	2.52
48	0.153	0.164	1.67	1.69
72	0.068	0.083	0.92	1.00
96	0.039	0.046	0.55	0.55
Cmax (µg equiv/mL)	0.723	0.664	5.01	4.18
Cmax/D	0.682	0.582	0.503	0.418
tmax(hours)	8	6	12	4
t1/2  (hours)	24.6	26.2	30.0	29.3
AUC(0-t)    (µg equiv.h/g)	22.2	23.6	198	179
AUC(0-t)/D	20.9	20.7	19.9	17.9
AUC(0-inf)   (µg equiv.h/g)	23.6	25.4	222	202
AUC(0-inf)/D	22.2	22.3	22.2	20.2
AUC % Extrap	5.94	6.85	10.8	11.4

PK parameters are Phoenix (WinNonlin) derived.

Note: Dose normalised parameters were calculated using mean actual doses.

Table 5: Mean Blood Concentrations and Pharmacokinetic Parameters Following Single Intravenous Administration of [14C]-Stest substance to Rats Expressed as µg Equivalents of test substance / g

Nominal Time after dosing (h)	Group 3 – 1 mg/kg iv
	Male	Female
0.25	0.290	0.282
1	0.247	0.217
4	0.281	0.240
8	0.304	0.250
12	0.298	0.282
24	0.282	0.288
48	0.160	0.170
72	0.107	0.103
C0 (µg equiv/g)	0.306	0.308
t1/2(hours)	35.0*	25.7
AUC(0-t) (µg equiv.h/g)	15.4	15.2
AUC(0-t)/D	15.1	14.6
AUC(0-inf)(µg equiv.h/g)	20.8*	16.7
AUC(0-inf)/D	20.4*	16.1
AUC % Extrap	26.0*	17.5
MRT(0-inf)(h)	53.2*	42.8
CL (g/h/kg)	49.2*	62.1
Vss (g/kg)	2618*	2660

PK parameters are Phoenix (WinNonlin) derived

* = The extrapolation of the AUC to infinity represents more than 20 % of the total area

Table 6 Mean Blood to Plasma Ratios Following Single Oral Administration of [14C]-test substance to Rats

Nominal Time after dosing (h)	Group 1 - 1 mg/kg		Group 2 – 10 mg/kg
	Male	Female	Male	Female
0.5	0.5	0.5	0.6	0.6
2	0.5	0.5	0.6	0.6
4	0.6	0.6	0.6	0.6
6	0.6	0.6	0.5	0.6
8	0.6	0.6	0.6	0.6
12	0.6	0.5	0.5	0.6
24	0.7	0.7	0.6	0.7
48	0.8	0.8	0.8	0.8
72	1.1	1.1	0.9	0.9
96	1.2	1.3	1.0	1.1

Table 7 Relationship between AUC(0-t) and Cmax of Total Radioactivity in Blood and Plasma Following a Single Oral Administration of [14C]-test substance to Rats (Groups 1 & 2)

Blood:

Males
Nominal Dose	Fold Increase	Cmax (µg equiv/g)	Fold Increase	AUC(0-t) (µg equiv.h/g)	Fold Increase
1	-	0.411	-	15.3	-
10	10	2.71	6.6	132	8.6
Females
Nominal Dose	Fold Increase	Cmax (µg equiv/g)	Fold Increase	AUC(0-t) (µg equiv.h/g)	Fold Increase
1	-	0.418	-	16.6	-
10	10	2.60	6.2	128	7.7

 Plasma: 

Males
Nominal Dose	Fold Increase	Cmax (µg equiv/mL)	Fold Increase	AUC(0-t) (µg equiv.h/mL)	Fold Increase
1	-	0.723	-	22.2	-
10	10	5.01	6.9	198	8.9
Females
Nominal Dose	Fold Increase	Cmax (µg equiv/mL)	Fold Increase	AUC(0-t) (µg equiv.h/mL)	Fold Increase
1	-	0.664	-	23.6	-
10	10	4.18	6.3	179	7.6

Conclusions:

The absorption and systemic availability of total radioactivity was > 88 % following oral administration of [14C]-test substance to rats, irrespective of dose and sex. The extent of systemic exposure was sub-proportional between the 1 and 10 mg/kg doses. Total radioactivity remained predominantly in the plasma fraction, with the association decreasing at later time points. In general, there were no consistent sex-related differences noted in the pharmacokinetics of total radioactivity

Executive summary:

Single oral doses of 1 and 10 mg/kg and a single intravenous (iv) dose of 1 mg/kg [14C]-test substance were administered to subgroups (oral) or groups (iv) of 4 male and 4 female animals. Blood samples were taken over a 4 (oral) or 3 (iv) day period to determine the pharmacokinetics of total radioactivity in blood (following oral and iv administration) and plasma (following oral administration). Oral bioavailability was determined by comparing the dose normalised exposures following oral and iv administration of [14C]-test substance. The nature and identity of metabolites present in the plasma from oral groups were also investigated and reported separately.

Following a single oral administration of 1 mg/kg [14C]-test substance, peak blood and plasma concentrations were observed at 6-8 hours post dose. At the higher dose (10 mg/kg) the tmax was observed at 12 and 4 hours post dose, in males and females, respectively. Overall total systemic exposure was greater in plasma than whole blood within the same dose levels, being more pronounced at the higher dose (10 mg/kg). Systemic exposure to total radioactivity (based on Cmax and AUC(0-t) estimates) increased in a sub-proportional manner between the 1 and 10 mg/kg dose levels in whole blood and plasma for both males and females. The absolute bioavailability was estimated to be 96-100 % at the low dose suggesting absorption was complete, however at the high dose absolute bioavailability was estimated to be 88% indicating absorption was not complete. In general, there were no consistent sex-related differences noted in the pharmacokinetics of total radioactivity.

In the oral dose groups, blood to plasma ratios suggested that total radioactivity remained predominantly in plasma rather than the cellular component of whole blood at earlier time points. The blood to plasma ratio appeared to increase at later time points, becoming either evenly distributed or greater in the cellular fraction.

Following a single intravenous dose of 1 mg/kg [14C]-test substance to male and female rats, the concentration of radioactivity in blood plateaued to 24 hours post dose, then steadily declined to 72 hours post dose. The systemic exposure (AUC(0-t)) was comparable irrespective of sex at 15.2 - 15.4 µg equiv.h/g. In males, an apparent terminal phase could not be reliably estimated.

The absorption and systemic availability of total radioactivity was >88% following oral administration of [14C]-test substance to rats, irrespective of dose and sex. The extent of systemic exposure was sub-proportional between the 1 and 10 mg/kg doses. Total radioactivity remained predominantly in the plasma fraction, with the association decreasing at later time points. In general, there were no consistent sex-related differences noted in the pharmacokinetics of total radioactivity.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

July 2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

other: EC 1107/2009

Version / remarks:

October 2009

Qualifier:

according to guideline

Guideline:

other: EU 283/2013

Version / remarks:

March 2013

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 Nohsan No 8147

Version / remarks:

November 2000

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

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 and 2: 8 - 9 weeks; Group 3: 8 - 9 weeks (at time of first dose)
- Weight at study initiation: 222 - 310 g and 155 - 202 g for males and females, respectively (Group 1); 227 - 284 g and 160 - 187 g for males and females, respectively (Group 2), 225 - 260 g for males (Group 3, at the time of first dose).
- Housing: Pre-study: multiply housed by sex in solid bottomed polycarbonate cages with wood shavings. On study: Multiply housed by sex in polycarbonate and stainless steel cages with raised wire mesh floors. Rats used for excretion were returned to pre-study housing following day 7 excreta collections. During excreta collection period’s rats were housed singly in all-glass metabolism cages.
- Diet: A standard laboratory diet of known formulation, ad libitum.
- Water: mains tap water, ad libitum
- Acclimatisation: At least 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 17 - 22
- Humidity (%): 29 - 61
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

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:
All dose preparations were stored in a fridge set to maintain 4 °C when not in use.
For the Group 1 preparation, an accurate volume of [14C]-test substance stock solution was transferred into a mortar bowl. For Group 2 and 3 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 transferred 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. If necessary, a spatula was used to aid removal of the [14C]-test substance from the mortar bowl. The remaining [14C]-test substance was lightly wetted with an appropriate amount of dose vehicle (0.5 % aqueous CMC containing 0.5 % Tween 80) 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 and 7 or 14 daily oral doses

Dose / conc.:

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

Remarks:

Group 1 - single oral dose of [14C]-test substance

Dose / conc.:

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

Remarks:

Group 2 - single oral dose of [14C]-test substance

Dose / conc.:

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

Remarks:

Group 3 - daily doses for 7 consecutive days of [14C]-test substance

No. of animals per sex per dose / concentration:

20 for Group 1 and 2, and 12 males for Group 3.

Control animals:

no

Details on study design:

Groups of 40 male and 40 female rats were each given a single oral dose of [14C]-test substance at nominally 1 mg/kg (Group 1) or 10 mg/kg (Group 2) or a group of 12 male rats were given a multiple oral daily dose (up to 14 consecutive doses) of
[14C]-test substance at nominally 10 mg/kg/day (Group 3). In each group selected organs/tissues and blood were taken at 5 (single dose) or 3 (multiple dose) predetermined time points and concentrations of radioactivity determined.

Details on dosing and sampling:

DOSE ADMINISTRATION
Each animal was accurately weighed prior to dosing. The syringes for oral administration 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 specific activity of the test substance. One male animale refluxed dose on dosing day 9. The excretion and tissue depletion profile were consistent within the group suggesting the reflux had minimal impact on the data. The dose preparations were administered by metal gastric gavage at 5 mL/kg, to achieve target doses of 1 mg/kg (Group 1), 10 mg/kg (Group 2) and 10 mg/kg/day (Group 3). Group 3 four male animals received 7 consecutive daily doses, 8 male animales received 14 consecutive daily.

CLINICAL OBSERVATIONS
Animals were observed at least twice daily on study.

COLLECTION OF EXCRETA
A number of animals were additionally used for excreta collection. Following dosing on days 1 and 7, urine and faeces were frozen upon excretion over solid carbon dioxide and collected up to 24 hours post dose. Following dosing on day 14, urine and faeces were frozen upon excretion by collection over solid carbon dioxide (at 24 hour intervals up to 240 hours post dose 14). The cages were rinsed with ca 5 mL of water prior to the removal of the urine pot. At each collection timepoint 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.

TERMINATION
Termination time points were chosen based on pharmacokinetic data from previous study, with the first time point of the single dose around the Cmax in blood. Each animal was humanely killed by CO2 narcosis. A terminal blood sample was taken into heparinised tubes and a small volume (ca 0.5 mL) retained for radioanalysis. The remaining blood sample was centrifuged to obtain plasma. The gastrointestinal tract (and contents) and residual carcasses were retained. The following tissues were also removed from all animals: 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 were stored in a freezer set to maintain -20 °C prior to analysis except the remaining carcass and the Group 1 72 h liver samples. The remaining carcass was stored at ambient temperature once digestion solution was added. The Group 1 liver samples were stored at ambient temperature overnight before being transferred, pre and post analysis, to a freezer set to maintain -20 °C. Total radioactivity was determined in each sample collected.

ANALYTICAL TECHNIQUES
Radioactivity in all samples was quantified by LSC (direct analysis or following sample oxidation).

Type:

distribution

Results:

Tissue distribution was broadly similar between the sexes. Highest concentrations of radioactivity observed in the liver and kidney

Type:

excretion

Results:

The major route of elimination via the faeces over the 24 h collection period, with a relatively minor proportion of the dose excreted in the urine.

Details on distribution in tissues:

Group1: In male and female rats, the maximum blood (ca. 0.3 µg equiv/g) and plasma (ca. 0.5 µg equiv/mL) concentrations of total radioactivity were observed at 8 hours post dose. The highest tissue concentrations were present in the liver (≤ 3.753 µg equiv/g) and kidneys (≤ 2.919 µg equiv/g). The majority of tissue concentrations were above the levels of circulating radioactivity, with the exception of bone mineral and testes in males and brain in males and females. Tissue to blood concentration ratios ranged from 0.6 (male rat brain) to 11.2 (female rat liver) at 8 hours post dose. The radioactivity present in the tissues, gastrointestinal tract contents and residual carcass accounted for up to 94 % of the administered dose at 8 hours post dose. The concentration of total radioactivity in all tissues then declined steadily to 168 h post dose. In blood and plasma the concentration was ≤0.034 and 0.023 µg equiv/g or mL, respectively. The highest tissue concentrations were present in the kidneys (≤ 0.586 µg equiv/g). At 168 h all tissue concentrations (≤ 0.352 µg equiv/g) were above that of circulating radioactivity. By 168 hours post dose, the tissue to blood ratios ranged from 1.6 (male rat bone mineral and muscle) to 17.1 (male and female rat kidneys). Circulating concentrations of total radioactivity were more associated with the plasma fraction to 24 hours, were approximately equally distributed at 72 h then were associated with the cellular fraction at later time points to 168 hours post dose. The radioactivity present in the tissues, gastrointestinal tract contents and residual carcass accounted for up to 12% of the administered dose by 168 hours post dose.
Group 2: In male and female rats, the maximum blood (2.42 - 2.81 µg equiv/g) and plasma (ca. 3.7 µg equiv/mL) concentrations of total radioactivity were observed at 4 hours post dose. The highest tissue concentrations were present in the liver (≤2 4.27 µg equiv/g) and adrenals (≤ 25.01 µg equiv/g). The majority of tissue concentrations were above the levels of circulating radioactivity, with the exception of the brain in males. Tissue to blood concentration ratios ranged from 1.0 (male rat brain) to 10.9 (female rat adrenals) at 4 hours post dose. The radioactivity present in the tissues, gastrointestinal tract contents and residual carcass accounted for up to 91 % of the administered dose at 4 hours post dose. The concentration of total radioactivity in all tissues then declined steadily to 192 h post dose. In blood and plasma the concentration was ≤ 0.28 and 0.14 µg equiv/g or mL, respectively. The highest tissue concentrations were present in the kidneys (≤ 4.48 µg equiv/g). At 192 h all tissue concentrations (≤ 2.52 µg equiv/g) were above that of circulating radioactivity. By 192 hours post dose, the tissue to blood ratios ranged from 1.5 (male rat muscle and female rat bone) to 18.8 (male rat kidneys). Circulating concentrations of total radioactivity were more associated with the plasma fraction to 72 hours, then were associated with the cellular fraction at later time points to 192 hours post dose. The radioactivity present in the tissues, gastrointestinal tract contents and residual carcass accounted for up to 8.8% of the administered dose by 192 hours post dose.
Group 3: Dose related radioactivity was detectable in each tissue at the first sampling time point of 24 hours after the seventh dose. The highest mean tissue concentrations of radioactivity were observed at 24 hours after the fourteenth and final dose. Maximum blood and plasma concentrations of total radioactivity were 6.59 and 7.30 µg equiv/g or mL, respectively. The highest tissue concentrations were observed in the kidneys and liver with means of 88.57 and 46.27 µg equiv/g, respectively. All tissue concentrations were above the levels of circulating radioactivity (7.87 - 38.49 µg equiv/g). Following Cmax the concentration of total radioactivity in all tissues declined to 10 days after the fourteenth dose, where the concentrations of circulating radioactivity in blood and plasma were 2.09 and 0.38 µg equiv/g or mL, respectively. The highest tissue concentrations were observed in the kidneys and spleen with means of 31.89 and 19.84 µg equiv/g, respectively. At this final time point all tissue concentrations (excluding muscle) were above that of circulating radioactivity (2.53 - 12.34 µg equiv/g). Tissue to blood concentration ratios ranged from 1.2 (brain) to 12.1 (kidneys) at 24 hours after the seventh dose. By 10 days after the fourteenth dose, the tissue to blood ratios ranged from 1.0 (muscle) to 15.4 (kidneys). Circulating concentrations of total radioactivity were initially more associated with the plasma fraction up to 24 hours after the fourteenth dose then were associated with the cellular fraction at the final sampling point of 10 days after the fourteenth dose. Radioactivity present in the tissues, gastrointestinal tract contents and residual carcass accounted for 24% of the administered dose at 24 hours after the seventh dose, declining to 2.9 % by 10 days after the fourteenth dose.

Details on excretion:

The apparent terminal phase half-life (t½) for tissue depletion of total radioactivity associated with [14C]-test substance in various tissues was characterised using mean tissue concentration vs time data derived from 4 animals per time point. The terminal phase half-life estimates for tissue depletion of total radioactivity in plasma was ca 34 - 39 hours following the 1 mg/kg dose and 10 mg/kg dose. However, longer half-life estimates were obtained for whole blood (51 - 79 hours for 1 mg/kg dose, 68 - 78 hours for 10 mg/kg dose). The calculated half-lives for tissue depletion were variable with the shortest estimate obtained for plasma and longest for pancreas being 34 and 91 hours, respectively.
- Following the first oral dose, the major route of elimination was via the faeces (26 %) over the 24 h collection period with 2.2 % of the administered radioactivity recovered in the urine. Recovery of administered radioactivity was incomplete with a mean of 29 % of the dose (including cage wash) excreted over this period.
- Following 7 repeated daily oral doses, the major route of elimination was via the faeces (14 %) over the 24 h collection period with 0.9 % of the administered radioactivity recovered in the urine. Recovery of administered radioactivity was incomplete with a mean of 1 5% of the dose (including cage wash) excreted over this period.
- Following 14 repeated daily oral doses, the major route of elimination was via the faeces, with 25 % of the administered radioactivity recovered over the entire collection period. Urinary elimination accounted for 1.1 % of the dose. An additional 0.6 % of the dose was recovered in cage washes.
- Excretion was nearing completion 10 days after the fourteenth dose with 2.9 % remaining in carcass, tissues and gastrointestinal tract. The total mean recovery of administered radioactivity including excreta, cage wash, tissues, gastrointestinal tract and residual carcass was 30 %. The total recovery represents the selected sampling points only and is not representative of a quantitative recovery.

Metabolites identified:

no

ANALYSIS OF DOSE PREPARATION

Prior to dose preparation the radiochemical was shown to have a purity of > 97 % as determined by HPLC and TLC. The pre- and post-dose radiochemical purities of [14C]-test substance were > 95 %, as determined by HPLC and TLC, indicating that the test item was 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 0.8 - 4.6 % of the mean indicating that a satisfactory homogeneity had been achieved.

ANIMAL OBSERVATIONS

No adverse animal observations were noted in this study.

Table 2: Distribution of radioactivity in tissues/organs 8, 24, 72, 120 and 168 hour safter single oral administration of [14C]‑test substance to male rats at a dose of 1 mg/kg

	Group mean tissue residues (µg equiv/g or mL)
Time after dosing	8 h	24 h	72 h	120 h	168 h
Adrenals	2.212	1.154	0.471	0.272	0.242
Bone Mineral	0.334	0.291	0.120	0.057	0.050
Brain	0.191	0.165	0.122	0.073	0.061
Fat-Renal	1.323	1.026	0.273	0.111	0.084
Heart	1.067	0.572	0.213	0.104	0.142
Kidneys	2.919	2.365	1.284	0.649	0.536
Liver	3.753	1.994	0.663	0.278	0.224
Lungs	1.027	0.658	0.309	0.157	0.152
Muscle	0.511	0.293	0.113	0.057	0.050
Pancreas	1.455	0.915	0.433	0.204	0.179
Plasma	0.534	0.322	0.091	0.024	0.019
Spleen	0.765	0.635	0.431	0.279	0.297
Testes	0.326	0.254	0.138	0.075	0.061
Thymus	0.714	0.549	0.326	0.196	0.194
Thyroid	1.507	0.728	0.299	0.161	0.158
Whole blood	0.351	0.210	0.084	0.037	0.031

Table 3: Distribution of radioactivity in tissues/organs 8, 24, 72, 120 and 168 hours after single oral administration of [14C]-test substance to female rats at a dose of 1 mg/kg

	Group mean tissue residues (µg equiv/g or mL)
Time after dosing	8 h	24 h	72 h	120 h	168 h
Adrenals	2.047	1.299	0.600	0.422	0.352
Bone Mineral	0.350	0.278	0.108	0.072	0.066
Brain	0.220	0.203	0.129	0.110	0.085
Fat-Renal	1.994	1.130	0.285	0.144	0.109
Heart	1.067	0.694	0.224	0.148	0.117
Kidneys	2.401	2.191	1.025	0.739	0.586
Liver	3.560	2.167	0.636	0.429	0.345
Lungs	1.091	0.750	0.335	0.232	0.196
Muscle	0.535	0.344	0.118	0.080	0.068
Ovaries	1.149	0.685	0.415	0.370	0.289
Pancreas	1.600	0.950	0.451	0.315	0.243
Plasma	0.521	0.398	0.078	0.030	0.023
Spleen	0.823	0.811	0.460	0.380	0.327
Thymus	0.729	0.594	0.335	0.270	0.235
Thyroid	1.098	0.752	0.328	0.184	0.188
Uterus	0.715	0.720	0.247	0.196	0.160
Whole blood	0.318	0.248	0.079	0.053	0.034

Table 4: Distribution of radioactivity in tissues/organs 4, 24, 72, 144 and 196 hours after single oral administration of [14C]test substance to male rats at a dose of 10 mg/kg

	Group mean tissue residues (µg equiv/g or mL)
Time after dosing	4 h	24 h	72 h	144 h	192 h
Adrenals	19.80	11.71	4.37	2.32	1.76
Bone Mineral	2.90	1.99	1.21	0.45	0.39
Brain	2.60	1.42	1.05	0.57	0.47
Fat-Renal	19.33	17.76	3.06	1.08	0.69
Heart	8.82	5.58	2.12	0.95	0.66
Kidneys	14.61	18.46	12.05	5.62	4.48
Liver	24.27	15.14	6.13	2.65	1.68
Lungs	8.44	6.04	2.56	1.26	1.04
Muscle	4.89	2.82	1.07	0.51	0.36
Pancreas	13.60	8.95	3.70	1.90	1.33
Plasma	3.74	3.03	1.00	0.26	0.12
Spleen	5.46	4.70	3.86	2.49	2.13
Testes	3.16	2.14	1.25	0.63	0.46
Thymus	6.06	4.49	2.67	1.75	1.39
Thyroid	12.57	7.67	3.01	1.61	1.07
Whole blood	2.81	1.86	0.81	0.38	0.24

Table 5: Distribution of radioactivity in tissues/organs 4, 24, 72, 144 and 192 hours after single oral administration of [14C]test substance to female rats at a dose of 10 mg/kg

	Group mean tissue residues (µg equiv/g or mL)
Time after dosing	4 h	24 h	72 h	144 h	192 h
Adrenals	25.01	13.88	5.39	3.37	2.47
Bone Mineral	2.86	2.17	0.99	0.61	0.41
Brain	3.44	1.85	1.19	0.92	0.60
Fat-Renal	20.82	22.73	4.27	1.40	0.80
Heart	10.01	6.18	2.29	1.21	0.78
Kidneys	14.45	15.57	9.42	5.52	3.92
Liver	22.09	15.58	5.71	3.30	2.15
Lungs	9.07	6.37	3.10	1.91	1.34
Muscle	5.40	3.01	1.23	0.70	0.46
Ovaries	11.80	7.98	3.82	3.51	2.23
Pancreas	16.27	10.00	4.16	2.61	1.64
Plasma	3.65	3.20	0.94	0.23	0.14
Spleen	6.45	5.64	3.86	3.24	2.52
Thymus	6.99	5.05	2.82	2.23	1.72
Thyroid	12.25	7.49	3.23	2.24	1.20
Uterus	5.79	5.79	2.34	1.84	1.28
Whole blood	2.42	1.87	0.80	0.39	0.28

Table 6:  Distribution of radioactivity in tissues/organs 24 hours post dose 7, 24 hours and 10 days post dose 14 after a repeated daily oral dose oral administration of [14C] test substance to male rats at a dose of 10 mg/kg/day

	Group mean tissue residues (µg equiv/g or mL)
Time after dosing	24 h pd 7	24 h pd 14	10 days pd 14
Adrenals	30.50	38.49	12.16
Bone Mineral	6.57	8.33	3.00
Brain	6.05	7.87	3.08
Fat-Renal	46.01	36.96	2.53
Heart	15.46	17.17	3.64
Kidneys	61.76	88.57	31.89
Liver	39.29	46.27	7.97
Lungs	16.09	21.38	7.31
Muscle	7.90	9.00	2.02
Pancreas	25.90	30.12	7.66
Plasma	6.87	7.30	0.38
Spleen	20.99	32.16	19.84
Testes	7.49	8.91	2.76
Thymus	17.24	25.28	12.34
Thyroid	19.84	25.08	7.42
Whole blood	5.10	6.59	2.09

pd = Post dose

Table 7: Recovery of radioactivity in excreta and tissues after administration of a repeated daily oral dose of [14C]-test substance to rats

Time after dosing (hours)	Male (n=4)
	Urine	Faeces	Cage wash
0-24 (pd 1)	0.2 (2.2)	1.9 (26)	<0.1 (0.3)
0-24 (pd 7)	0.3 (0.9)	5.1 (14)	0.1 (0.2)
0-24 (pd 14)	0.3	7.4	0.1
24-48 (pd 14)	0.1	3.9	0.1
48-72 (pd 14)	0.1	2.5	0.2
72-96 (pd 14)	0.1	1.4	0.1
96-120 (pd 14)	<0.1	1.0	<0.1
120-144 (pd 14)	<0.1	0.6	<0.1
144-168 (pd 14)	<0.1	0.5	<0.1
168-192 (pd 14)	<0.1	0.4	<0.1
192-216 (pd 14)	<0.1	0.3	<0.1
216-240 (pd 14)	<0.1	0.3	<0.1
Subtotal	1.1	25	0.6
Tissues	0.6
GI tract	0.2
GI tract contents	0.1
Carcass	2.1
Total Recovery^A	30

pd = Post dose

A = The total excretion and recovery represent the selected sampling points only and are not representative of a quantitative elimination or overall recovery.

Note: The % recovery presented is the overall recovery following 14 dose administrations. The % recovery presented in ( ) is the recovery following 1 or 7 respective dose administrations. 

Table 8: Elimination of radioactivity from rat tissues/organs after single administration of [14C]test substance to rats at a dose of 1 or 10 mg/kg

	Values are expressed ast ½el (h). Each value is a mean of 4 rats
	Male		Female
Tissue	1 mg/kg	10 mg/kg	1 mg/kg	10 mg/kg
Adrenals	63.6	90.3	125^#	107^#
Bone Mineral	55.2	67.1	70.5^#	95.0^#
Brain	95.1^#	101^#	160^#	111^#
Fat-Renal	39.6	55.1	69.2	49.2
Heart	67.9^#	70.5	102^#	77.3
Kidneys	64.8	82.2^#	119^#	94.8^#
Liver	44.8	63.9	109^#	85.6
Lungs	65.6	90.1	124^#	99.5^#
Muscle	55.6	75.6	121^#	84.9
Ovaries	NA	NA	184^#	105^#
Pancreas	58.9	80.7	108^#	90.5
Plasma	33.9	39.0	35.0	36.9
Spleen	123^#	141^#	195^#	157^#
Testes	68.1^#	75.3	NA	NA
Thymus	91.7^#	127^#	188^#	171^#
Thyroid	64.0	80.4	70.2^#	69.3
Uterus	NA	NA	153^#	142^#
Whole Blood	50.7	68.2	78.9	78.4

NA = Not applicable

# = The coefficient of determination of the elimination phase was < 0.800 and/or extrapolation of the AUC to infinity represents more than 20 % of the total area.

Conclusions:

Following single and repeat oral dosing of [14C]-test substance, dose related radioactivity was distributed extensively to all tissues. The highest concentrations of radioactivity were generally observed in the liver and kidney, consistent with the excretion profile of [14C]-test substance. The tissue distribution and depletion of radioactivity was broadly similar, irrespective of dose and sex. Following a single 1 mg/kg or 10 mg/kg oral dose, total tissue and carcass residues at the last sampling point accounted for < 12 % of the administered dose. Ten days after 14 daily oral administrations of 10 mg/kg [14C] test substance, 2.9 % of the dose remained in the carcass, tissues and gastrointestinal tract, indicating excretion was nearing completion.

Executive summary:

To investigate the tissue distribution and depletion of dose-related radioactivity, two groups of 40 Han Wistar rats (20 per sex) were given a single oral dose of 1 and 10 mg/kg [14C]-test substance in a GLP-compliant OECD 417 study. In addition, one group of 12 male rats was given single oral doses of 10 mg/kg/day [14C]-test substance for up to 14 consecutive days. Following dosing, groups of 4 rats per sex per time point were humanely killed at time points chosen using pharmacokinetic data from another study. The first time point chosen, following single oral administration, was based around the Cmax observed in blood. For the 1 mg/kg single dose groups, 8, 24, 72, 120 and 168 h were chosen. For the 10 mg/kg single dose group, these were 4, 24, 72, 144 and 192 h. For the repeat dose group, rats were killed at 24 h post the 7th dose and 24 h and 10 days post the 14th dose. Residual radioactivity was measured in selected tissues/organs and the remaining carcasses. Where appropriate, terminal phase half lives of depletion were calculated for individual tissues from animals in Groups 1 and 2. Additionally, the excretion of radioactivity in urine and faeces was monitored in one group of multiply dosed rats for a period of 24 h following the first and seventh doses, then at 24 h intervals following the fourteenth dose until 240 h post dose 14. The nature and identity of metabolites present in selected plasma and excreta samples were also investigated and reported separately.

Following single oral administration of [14C]-test substance at 1 mg/kg, radioactivity was widely distributed to the tissues in both sexes. Peak mean tissue concentrations were observed at the first sampling time of 8 hours post dose in males and females. The highest mean tissue concentrations in males and females were obtained in the liver (3.753 and 3.560 µg equiv/g) and kidneys (2.919 and 2.401 µg equiv/g), respectively. The majority of other tissue concentrations were above that of circulating blood concentrations (0.351 and 0.318 µg equiv/g in males and females, respectively). The liver and kidney concentrations are consistent with both biliary and urinary elimination. Thereafter, all tissue concentrations declined steadily up to 168 hours post dose. All tissue concentrations were above that of circulating blood (0.031 and 0.034 µg equiv/g in males and females, respectively). The total mean residues in tissues and carcass at 168 hours accounted for 11 % and 12 % of the dose in males and females, respectively.

Following single oral administration of [14C]-test substance at 10 mg/kg, radioactivity was widely distributed to the tissues in both sexes. Peak mean tissue concentrations were attained at the first sampling time of 4 hours post dose. The highest mean tissue concentrations were observed in the liver (24.27 and 22.09 µg equiv/g) and adrenals (19.80 and 25.01 µg equiv/g), in males and females, respectively. All tissue concentrations (excluding male brain) were above that of circulating blood concentrations (2.81 and 2.42 µg equiv/g in males and females, respectively). Thereafter, all tissue concentrations declined steadily up to 192 hours post dose. All tissue concentrations were above that of circulating blood (0.24 and 0.28 µg equiv/g in males and females, respectively). The total mean residues in tissues and carcass at 192 hours accounted for 7.1 % and 8.8 % of the dose in males and females, respectively.

Following repeated daily oral administration of [14C]-test substance at 10 mg/kg to male rats for up to 14 consecutive days, radioactivity was widely distributed to the tissues. Mean concentrations of total radioactivity in each tissue were detectable at the first sampling time point of 24 hours after the seventh dose. The highest mean tissue concentrations of radioactivity were observed at 24 hours after the fourteenth and final dose. The highest tissue concentrations were observed in the kidneys and liver with means of 88.57 and 46.27 µg equiv/g, respectively. All tissue concentrations were above that of circulating blood concentrations (6.59 µg equiv/g). At the final sampling point of 10 days after the fourteenth dose, the concentration of total radioactivity in all tissues had declined. All tissue concentrations were above that of circulating blood (2.09 µg equiv/g in males and females, respectively), except for muscle, which was similar to the level of circulating blood. The total mean residues in tissues and carcass at 10 days post dose 14 accounted for 2.9 % of the dose.

Following a single dose and 7 or 14 daily oral doses, the major route of elimination was via the faeces, with a relatively minor proportion of the dose excreted in the urine. The repeated dosing, therefore, did not appear to change the excretion profile.

Throughout the course of the study, the majority of mean tissue concentrations were, in general, broadly similar between males and females at both doses. Consistent with this, estimates for tissue depletion half-life appeared similar in male animals and female animals, where reliable estimates were available.

The tissue depletion half-life of dose related radioactivity in the majority of tissues were either similar or longer than in whole blood and plasma. Whole blood estimates were longer than that obtained in plasma. Consistent with this, circulating concentrations of total radioactivity were initially more associated with the plasma fraction then generally became increasingly associated with the cellular fraction at later time points.

Following single and repeat oral dosing of [14C]-test substance, dose related radioactivity was distributed extensively to all tissues. The highest concentrations of radioactivity were generally observed in the liver and kidney, consistent with the excretion profile of [14C] test substance. The tissue distribution and depletion of radioactivity was broadly similar, irrespective of dose and sex. Following a single 1 mg/kg or 10 mg/kg oral dose, total tissue and carcass residues at the last sampling point accounted for < 12 % of the administered dose. Ten days after 14 daily oral administrations of 10 mg/kg [14C]-test substance 7, 2.9 % of the dose remained in the carcass, tissues and gastrointestinal tract, indicating excretion was nearing completion.

Description of key information

- Absorption: the absorption of total radioactivity was >88% following oral administration of [14C]-labelled test substance, irrespective of dose and sex.

- Distribution: the tissue distribution and depletion of radioactivity was broadly similar, irrespective of dose and sex and was distributed extensively to all tissues. The highest concentrations of radioactivity were generally observed in the liver and kidney.

- Metabolism: Metabolism of test substance was extensive. The metabolite profile was similar across all samples, irrespective of dose, single or repeat dose, radio-label and sex. The biotransformation resulted in ring opening of the isoxazole ring, ring opening and cleavage of the oxazolidinone ring, oxidative defluorination and glucuronic acid conjugation.  

- Excretion: The majority of the absorbed dose was excreted in faeces via biliary elimination.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

88

Absorption rate - inhalation (%):

100

Additional information

Absorption following oral administration

Following a single oral administration of [14C]-labelled test substance, the majority of dose related radioactivity was eliminated by 72 h post dose, but excretion was incomplete at 168 h, irrespective of dose, radiolabel or sex. Absorption of [methylphenyl-14C]-labelled 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]-labelled 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]-labelled test substance. The absorption and systemic availability of total radioactivity was >88% following oral administration of [14C]-labelled test substance to rats, irrespective of dose and sex. The extent of systemic exposure was sub-proportional between the 1 and 10 mg/kg doses. Total radioactivity remained predominantly in the plasma fraction, with the association decreasing at later time points. In general, there were no consistent sex-related differences noted in the pharmacokinetics of total radioactivity.

Distribution following oral administration

Following single and repeat oral dosing of [14C]-labelled test substance, dose related radioactivity was distributed extensively to all tissues. The highest concentrations of radioactivity were generally observed in the liver and kidney, consistent with the excretion profile of [14C]-labelled test substance. The tissue distribution and depletion of radioactivity was broadly similar, irrespective of dose and sex. Following a single 1 mg/kg or 10 mg/kg oral dose, total tissue and carcass residues at the last sampling point accounted for <12% of the administered dose. Ten days after 14 daily oral administrations of 10 mg/kg [14C]-labelled test substance, 2.9% of the dose remained in the carcass, tissues and gastrointestinal tract, indicating excretion was nearing completion.

Metabolism following oral administration

Metabolism of test substance was extensive with unchanged test substance a minor component in excreta less than 3.5% of the dose. In plasma, unchanged test substance accounted for 3.8 to 6.4% of the total radioactivity AUC (TRA) following a 1 mg/kg dose and 12.5 - 13.9% of the TRA following a 10 mg/kg dose. In general, the metabolite profile was similar across all samples, irrespective of dose, single or repeat dose, radio-label and sex.

Excretion following oral administration

The majority of administered dose (AD) was excreted via bile into faeces, with a limited fraction excreted in urine. M22 was the largest circulating metabolite at up to 80% of the TRA. M22 and its glucuronide accounted for up to 10% of the AD in bile. Other metabolites accounting for >5% AD in bile were M6 (6.0 - 11.0% dose) and M21 glucuronide (5.4% AD).

Absorption following dermal administration

The absorption of the substance through rat and human skin was studied in a "triple back" of in vivo and in vitro studies, performed under GLP to the relevant OECD test guidelines, using a representative soluble concentrate type formulation containing 200 g test substance/L. All studies used an exposure period of 10 hours, followed by a thorough skin wash. The same test concentrations of 200 g/L (undiluted formulation), 7.5, 2 and 0.2 g/L (applied as aqueous dilutions) were used in the tests. The total observation period was 24 hours in the two in vitro studies using rat and human split-thickness skin membranes. For the in vivo study of dermal absorption in the rat animals were sacrificed at 10, 24, 72 and 120 hours following the removal of test substance by the skin wash. For all studies, tape stripping was done and the first two tape strips were considered unabsorbed. The dermal absorption of substance through rat split-thickness skin membranes over a period of 24 hours was 0.02% for the formulation concentrate (200 g/L) and ranged from 0.35%, 0.57% to 1.54% for spray dilutions (7.5, 2.0 and 0.2 g/L). Following in vivo exposure of rat skin to radiolabelled substance for a period of 10 hours, 2.15%, 18%, 14% and 3.56% of applied dose was absorbed over a 120 hour post dose period in the groups treated with the formulation concentrate (200 g/L), or 1/26.7 (7.5 g/L), 1/100 (2.0 g/L) and 1/1000 (0.2 g/L) aqueous dilutions, respectively. Clearly lower absorption was observed in the in vitro studies with human split-thickness skin. The dermal absorption of substance through human split-thickness skin membranes over a period of 24 hours was 0.01% for the formulation concentrate (200 g/L) and ranged from 0.02%, 0.09% to 0.18% for spray dilutions (7.5, 2.0 and 0.2 g/L). The likely in vivo absorption through human skin was estimated by using the absorption values determined in the three studies: % in vivo human absorption = % in vivo rat absorption / (% in vitro rat absorption / % in vitro human absorption). Applying the experimental data, the in vivo absorption value through human skin is 0.09% for the undiluted formulation and 4.8% for aqueous dilutions (based on the highest absorption obtained at the concentration of 7.5 g/L).