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Description of key information

Absorption

Oral absorption was measured in the two biliary excretion studies. With the F344 rat, absorption was about 70% for both males and females at both low and high doses. With the Wistar rat, absorption was about 57% for both males and females at both low and high doses.

Dermal absorption in the rat increased with decreasing test item concentration and ranged between 0.37% (concentrate) to 5.55% (highest dilution (1:200)). No experimental data on Inhalation absorption is available.

Metabolism

The test substance is intensively metabolised. The main metabolic reactions are considered to be 1) hydrolytic elimination of both 3- and 4-cyclopropanecarbonyl groups, 2) hydroxylation at 4-methyl group, 3) N-oxidation of pyridine ring and 4) conjugation at hydroxyl-groups (glucuronidation).

Distribution

The test substance distributed rapidly to the tissues and Tmax occurred within 0.5 to 2 hours following administration (depending on dose level). There was widespread tissue distribution.

Excretion

Excretion occurred via urine and faces, with feces being the predominant route , and was nearly completed within 96 h – 120 h after treatment

No significant sex related difference was observed in toxicokinetics.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
57
Absorption rate - dermal (%):
5.55
Absorption rate - inhalation (%):
100

Additional information

WoE study: Pharmacokinetics 2016/1019953

This report outlines the results of investigations on the plasma kinetics as well as absorption, distribution, and elimination of 14C labelled test substance in female F344 rats. This was a mechanistic study conducted with the primary intent of measuring the pharmokinetic properties of the test substance in the strain and sex of rat as well as the doses that were relevant to the rat carcinogenicity studies conducted with the test substance.

Another important aspect of this study was that this study measured the pharmacokinetic properties of a major cyclopropanecarboxylic acid-related metabolite (CPCA-carnitine, M440I060) in the rat.

Plasma kinetics were investigated after 14 days of dietary intake followed with a single oral gavage administration of the radiolabeled test substance on Day 15. Target dose levels were 3, 15 and 50 mg/kg bw. At least 4 animals per dose group were included. Blood samples (approximately 300 μL) were taken on Day 1 [predose] and on Day 15 [predose, 1, 2, 4, 8, 24, 48 and 72 h after oral dosing]. The concentrations of the radioactive residues in plasma were analyzed by LSC.

In addition to measuring traditional PK parameters (related to total radioactivity measured in plasma – that does not allow to differentiation between parent compound and/or its metabolites), this study measured PK parameters in the plasma of four test substance-related residues. To obtain the maximum detection sensitivity as well as to accurately judge the impact of repeated dosing, analysis was conducted via LC/MS. The four residues analyzed were parent substance, M440I017, M440I001 and M440I060 (CPCA-Carnitine).

Urine and feces were collected in the same animals in 8-24, 24-48 and 48-72 hour time intervals. The time points for the sampling of organs / tissues selected for the tissue distribution experiments were based on the results of the plasma kinetics and corresponded to the time points of the maximum plasma concentrations. Rats were sacrificed at 1 and 1.5 h for respectively 15 and 50 mg/kg bw the test substance. After sacrifice, blood, liver and uterus were prepared and the radioactive residues of 14C labelled test substance were determined by LSC analyses after appropriate sample workups. These experiments allowed the evaluation of the distribution of the test substance into organs and tissues under the situation of defined plasma / blood concentrations.

In plasma kinetics, AUC values indicated an internal exposure that is clearly correlated to the dosing regimen of 14C labelled test substance. The AUC values of the test substance increased with increasing dose level in an un-proportional manner. From 3 to 15 mg/kg the AUC was increased 13.5x, and for a 17x increase in dose (3 to 50 mg/kg) the AUC increased 53x.

Matrix

 

Dose

(mg/kg bw)

Cmax

(ng Eq/g)

tmax

(h)

tlast

(h)

terminal

half-life

(h)

AUC0-last

(ng Eq*~h/g)

AUC0-∞

(ng Eq*h/g)

plasma

3, p.o.

262

1

24

8.17

728

800

15, p.o.

2800

1

24

5.19*

10400

10800

50, p.o.

6980

1-2$

24

4.21

41400

42300

blood

3, p.o.

221

1

72

96.5*

1600

3290*

15, p.o.

2110

1

72

55.2*

11700

15500*

50, p.o.

5050

1-2$

72

NR

43100

NR

* approximation, $range; NR not reported

In plasma kinetics of the test substance and its metabolites (bioanalytical data), the AUD values of the test substance increased with increasing dose level in an unproportional manner. From 3 to 15 mg/kg the test substance the AUC was increased 44x. And for a 17x increase in dose (3 to 50 mg/kg) the AUC0 -4 increased 199x. The test substance and the metabolites M440I001 and M440I017 displayed comparable concentration time curves, while the metabolite CPCA-Carnitine displayed a plateau level with a minor decrease after repeated administration.

Matrix

Dose

[mg/kg bw]

Cmax

[ng/mL)

tmax
[h]

tlast
[h]

terminal
half-life

[h]

AUC0-24
[ng*h/mL]

AUC0-
[ng*h/mL]

AUD

[ng*h/mL]

test substance

3, p.o.

24.7

1

4

n/c

n/c

n/c

104

15, p.o.

1500

1

24

2.71

4470

4480

4530

50, p.o.

4750

2

24

2.17

20600

20700

20700

M440I001

3, p.o.

n/c

n/c

n/c

n/c

n/c

n/c

72.5

15, p.o.

153

1

24

4.41

578

590

635

50, p.o.

457

2

24

3.59

2600

2630

2690

M440I0017

3, p.o.

3.91

1

4

n/c

n/c

n/c

78.2

15, p.o.

51.6

1

24

3.84

431

438

482

50, p.o.

175

2

24

3.78

1280

1300

1350

AS1574

CPCA-

carnitine

3, p.o.

195

1

72

36.1*

2780

6300*

4930

15, p.o.

1560

8

72

46.6*

22600

50200*

36200

50, p.o.

4190

8

72

27.1*

71400

149000*

122000

*: approximation; n/c could not be calculated

Over an observation period of about 72 hours, respectively 0.9 %, 1.3 % and 1.6 % of the dose at 3, 15, and 50 mg/kg was excreted via urine. Excretion via faeces was significantly higher and amounted for a period of about 72 hours to 85 %, 90 % and 65 % of the dose at respectively 3 mg/kg bw, 15 mg/kg bw and 50 mg/kg bw. The major part of faecal excretion occurred for all groups within 8-48 hours. Tissue sampling demonstrated distribution to the liver and, to a lesser extent, the uterus.

WoE study: Excretion balance 2015/8000182

To understand the metabolic fate of the test substance in rats, 14C-radiolabelled test substance ([NCA-14C]) was administrated to male and female rats at a nominal dose of either 3 or 300 mg/kg bw, four animals per experiment group, by a single oral gavage. This study consisted of two experiments: 1) excretion/mass balance and 2) biliary excretion. In addition, metabolites in urine, feces and bile were identified/characterized.

Following a single oral administration of [NCA-14C] labelled test substance to rats, the dosed radioactivity was readily excreted within 96 hr through the urine and feces with the total excretion rate of approximately 93 to 95 % of applied dose (AD). The urinary and fecal excretion was 5.5 to 20.4 %AD and 73.4 to 87.0 %AD, respectively, indicating that the fecal excretion was predominant route for elimination of the test substance in rats.

96 hr post dose

Percentage of the administered dose

3 mg/kg bw

300 mg/kg bw

Male

Female

Male

Female

Urine

7.2

5.5

20.4

19.4

Feces

88.5

87.0

73.4

75.4

Cage Wash

0.4

0.3

0.5

0.4

Total Excreted

93.1

92.9

94.2

95.2

GI tract and contents

0.1

0.3

0.1

0.1

Carcass

0.8

0.7

0.9

0.9

Total remaining in body

0.9

0.9

1.0

0.9

Total

94.0

93.8

95.3

96.1

In the biliary excretion experiment, the excreted radioactivity amounted to approximately 92 to 97 %AD at 48 hr post dose. Estimated absorption rate (at 48 hr post dose), which was calculated as the sum of the radioactivity found in the bile, urine and residual carcass, was determined to be 67 to 72 %. Absorption did not differ significantly between high and low dose.

The results indicate the biliary excretion significantly contributes to elimination of the orally administered test substance, and reveal that the fecal excretion via biliary excretion is the predominant elimination route of the test substance. While biliary excretion at the 300 mg/kg bw dose is reduced significantly, the urine excretion is higher at the 300 mg/kg bw dose.

48 hr post dose

Percentage of the administered dose

3 mg/kg bw

300 mg/kg bw

Male

Female

Male

Female

Bile

53.1

53.3

40.2

40.5

Urine

16.2

13.0

29.9

30.3

Feces

22.4

26.7

26.4

24.1

Cage Wash

0.6

0.2

0.7

1.0

Total Excreted

92.3

93.2

97.1

95.8

GI tract and contents

1.0

1.0

2.8

4.2

Carcass

1.0

0.9

1.1

1.3

Total remaining in body

2.0

2.0

3.9

5.5

Total

94.3

95.2

101.0

101.3

Absorbed

70.3

67.2

71.1

72.0

The main metabolic reactions of the test substance are considered to be 1) hydrolytic elimination of both 3- and 4-cyclopropanecarbonyl groups, 2) hydroxylation at 4-methyl group, and 3) N-oxidation of pyridine ring. The primary reaction likely to be hydrolytic elimination of 3-cyclopropanecarbonyl group, then further hydrolysis or hydroxylation. In addition, the N-oxidation of pyridine ring occurs followed by production of various hydrolytic products. No significant sex related difference was observed in excretion route and metabolic pathway of [NCA-14C] labelled test substance in rats.

WoE study: Pharmacokinetics 2015/8000181

The study investigated the pharmacokinetics and tissue distribution of 14C labelled test substance following a single oral dose of 3 or 300 mg/kg/bw to male and female rats. The radioactive dose was approximately 130 to 270 μCi/kg body weight.

In the pharmacokinetic pilot study, and the pharmacokinetic study, pharmacokinetic parameters were calculated from the whole blood, plasma, and red blood cell total radioactivity concentration data. In the pharmacokinetic study, generally, Tmax occurred between 0.25 and 1 hour following administration of 3 mg/kg, and between 2 – 4 hours following administration of 300 mg/kg; radioactivity was distributed between the plasma and the red blood cells. Elimination half-life in whole blood was 1 – 2.5 hours following administration of 3 mg/kg and 15.1 – 16 hours following administration of 300 mg/kg.

The test substance distributed rapidly to the tissues and Tmax occurred at 0.5 hours following administration of 3 mg/kg, and 2 hours (the first sampling point) following administration of 300 mg/kg. There was widespread tissue distribution with highest levels occurring in the GI tract and contents, liver, adrenals, kidney, urinary bladder (male only), pancreas, prostate, uterus, ovaries, spleen (females only), pituitary (females only), fat, mesenteric lymph nodes, heart, and lung. Levels were notably reduced after 96 hours. Tissues with the highest percent of administered dose at the 2- and 24-hour termination times were GI tract and contents, liver, and residual carcass.

WoE study: Pharmacokinetics 2014/1224518

Plasma kinetics was investigated after single, oral (p.o.) and intravenous (i.v.) administration. Target dose levels were 3, 30, 300 (p.o.) and 0.5 (i.v.) mg/kg bw.

In tissue distribution experiments, rats were dosed once orally and subgroups of 3 rats per observation time point and gender were sacrificed 1 h, 2 h, 3.5 h and 8 h (both genders) for the low dose of 3 mg/kg bw as well as 4 h, 12 h, 24 h and 36 h (males) and 4 h, 14 h, 20 h and 36 h (females) for the high dose group of 300 mg/kg bw.

A biliary excretion experiment was conducted with bile-duct cannulated rats. After regeneration from surgery, animals were dosed with 14C labeled test substance and were placed in metabolism cages in order to collect bile at 3 hour intervals as well as urine and feces in 24 hour intervals up to 72 hours.

14C labelled test substance was rapidly absorbed from the gastrointestinal tract after oral administration to male and female rats and was quickly excreted via urine and feces. Excretion was more pronounced via feces.

After multiple administration of the test substance (14 oral administrations with unlabeled test substance at 300 mg/kg bw and one oral administration with labeled 14C labelled test substance at 300 mg/kg bw), urinary excretion was slightly decreased compared to single dosing.

Investigations on plasmakinetics demonstrate a non-linear relationship for the internal doses (measured as AUCs) versus the oral dose with an overproportional increase being already present at a target dose level of 30 mg/kg bw. These results indicate potential saturation of kinetics at higher doses with a stronger effect in female rats.

Based on bile excretion experiments, absorption of 14C labelled test substance in male and female rats after single oral dosing was comparable for both dose levels tested and was calculated to be about 57 % and 60 % of dose for males and females at a dose level of 300 mg/kg bw and about 57 % of dose for both sexes at a dose level of 3 mg/kg bw.

WoE study: Metabolism 2011/1286178

Absorption, distribution, metabolism and excretion of test substance was investigated in male and female rats. The present studies were conducted according to OECD guideline 417 and were divided into two parts a biokinetics and a metabolism study. In the biokinetics study, the absorption, distribution, elimination and plasmakinetics of 14C labelled test substance were investigated in male and female Wistar rats.

In the metabolism study, the excretion and metabolism of the test substance was investigated in male and female rats after single, oral (by gavage) administration of 3 and 300 mg/kg bw and after repeated oral administration of (14 + 1) x 300 mg/kg bw 14C labelled test substance. Tissue and plasma samples were collected from male and female rats of dose groups V (3 mg/kg bw) and W (300 mg/kg bw). All animals of dose groups V and W were sacrificed 1 hour and 4 hours after treatment, respectively, and the respective samples were taken. In addition, urine, feces and bile samples from the biokinetics study after oral administration of the test substance were examined within the metabolism study.

Urine was collected from dose groups B, D and C after 6, 12 and 24 hours and subsequently in time intervals of 24 hours up to seven days. Feces from these dose groups was sampled in 24 hour intervals up to seven days. Bile was collected in time intervals of 3 hours for up to 72 hours and urine as well as feces was collected in 24 hour time intervals for 72 hours from male and female rats of dose groups R and S.

Excretion of radioactive residues

For all dose groups, excretion via urine, feces and bile was nearly completed within 96 h – 120 h after treatment. The major part of the radioactive residues was excreted via feces (72 % – 87 % of the dose within seven days) and lower portions were excreted via urine (5% – 21% of the dose). Route and rate of excretion of radioactive residues was similar for both sexes. Excretion was slower in the high dose groups compared to the low dose groups, and the portions excreted via urine were higher after administration of the high dose.

Analysis and identification of metabolites in matrices

Within the metabolism study, the nature of the biotransformation products of the test substance in excreta, bile, liver, kidney and plasma of rats after oral administration of the test item at two dose levels was investigated (3 and 300 mg/kg bw). Urine and feces samples (dose groups B, C and D) and bile samples (dose groups R and S) originating from the biokinetics study were used for the determination of the metabolic patterns. Two additional groups of animals (designated V and W) were treated specifically for the metabolism study.

Urine, bile, feces, liver, kidney and plasma were analyzed using HPLC with radio-detection. Identification was performed using HPLC-MS/MS and in some cases NMR. Metabolites present at 4% or greater of the administered dose were successfully identified. In addition, co-chromatography experiments and comparison of peak patterns were performed for assignment of further peaks and for confirmation.

Metabolite patterns in urine

The unchanged parent compound the test substance was detected in urine from rats of the dose groups BM, BF, DM, DF, CM, CF and SM in low portions (up to 0.26 % of the dose). A total of eleven metabolites was identified and quantified in the urine samples, and three glucuronic acid conjugates were detected in addition eluting together with their aglycones or an isomer thereof.

The main component in rat urine (except for dose group BF), metabolite M440I001, originates from the parent compound by the loss of both cyclopropane carboxylic acid (CPCA) ester moieties and can be conjugated with glucuronic acid at one of several hydroxyl groups (M440I054, not separated from M440I001). The most abundant component in urine from rats of dose group BF and second most abundant component in urine of the other dose groups (except for dose group BM) was metabolite M440I002, which is the respective derivative of the parent compound after loss of one CPCA ester moiety. In dose group BM the second most abundant component was M440I017, a pyridine N-oxidized derivative of parent.

Ten metabolites and one characterized component had lost one of the CPCA ester moieties, and four metabolites had cleaved both CPCA ester moieties.

The metabolite patterns in urine were qualitatively similar for both genders. Compared to urine of the low dose groups, the portions of the main component M440I001 / M440I054 and most other metabolites were considerably higher in urine of the high dose groups. Only the portion of the N-oxidized derivative M440I017 in urine of male rats was lower after single administration of the high dose. In urine of the repeated high dose group C, the portions of M440I002 / M=611 u and some other components were lower, and the portions of M440I008 / M440I043 and metabolite M440I039 were slightly higher compared to urine of the single high dose groups D.

Metabolite patterns in feces

The unchanged parent compound was identified as the main component in extracts of feces from rats of the low dose groups BM and BF in portions of 20.7 % and 36.7 % of the dose, respectively. In feces extracts of the dose groups DM, DF, CM and CF, the test substance accounting for the main portion was in a range from 5.3 % to 10.3 % of the dose, where the most abundant component was metabolite M440I001 followed by metabolite M440I058. A total of ten metabolites was identified and quantified in the feces extracts. No glucuronic acid conjugates or pyridine-N-oxidized derivatives were detected in feces.

Five metabolites had lost one of the CPCA ester moieties, and two metabolites and one characterized component had both CPCA esters cleaved.

The metabolite patterns in feces extracts were similar for both genders with a higher number of minor metabolites in feces extracts of dose groups BM and DM compared to female rats.

The relative portions of the parent compound in feces extracts from dose groups DM and DF were lower, and the portions of metabolites M440I058 and M440I008 were higher compared to extracts from feces of the low dose groups. After repeated administration of the high dose, the relative portions of metabolite M440I002 were lower in feces extracts of dose group C and the portions of metabolites M440I039 and M440I012 were slightly higher compared to dose group D.

Metabolite patterns in bile

The unchanged parent compound was detected in bile from rats of the dose groups RM, RF and SF in low portions and was not detected in bile of dose group SM. A total of twelve metabolites was identified and quantified in the bile samples.

The bile samples contained a notable number and high portions of pyridine-N-oxidized derivatives. The main component in rat bile, metabolite M440I017, is the pyridine-N-oxidized derivative of parent BAS 440 I. The second most abundant peak in bile represented metabolite M440I019 (the pyridine-N-oxidized derivative of M440I002) and a glucuronic acid conjugate of M440I058 (M440I059, not separated from M440I019).

Eight metabolites and one characterized component had lost one of the CPCA ester moieties, six metabolites and one characterized component had both CPCA esters cleaved.

The metabolite patterns in bile were qualitatively similar for both genders. Compared to bile of the low dose group R, the relative portions of the main component M440I017 and the parent compound were lower in sum in bile of high dose group S, and the portions of most other metabolites were higher after administration of the high dose.

Metabolite patterns in tissues and plasma

In the methanol extracts of liver and kidney and the supernatants obtained from plasma after protein precipitation, the main component was the test substance. The test substance was primarily accompanied by metabolites with lost CPCA ester moieties, M440I002 and M440I001 / M440I054, all below 1% of the dose.

Proposed metabolic pathway

The high number of identified metabolites and the low concentrations of the parent molecule, particularly in urine and bile, indicate intensive metabolism of the active substance. The main biotransformation reactions of the test substance in rats are as follows:

- hydrolytic loss of one or both CPCA ester moieties (ester cleavage)

- N-oxidation at the pyridine-ring

- hydroxylation of one of the methyl groups

- conjugation of hydroxyl groups with glucuronic acid

These main reactions or their combination produce a variety of metabolites. Further reactions are twofold hydroxylation of the parent molecule at a methyl group and at the pyridine-ring, complete oxidation of a methyl group to a carboxylic acid, dehydrogenation of a hydroxyl group, dehydration introducing an additional double bond or reduction of a hydroxyl group to hydrogen.

Key study: Dermal penetration 2014/1289878

The absorption, distribution and excretion of radioactivity of 14C labelled test substance was studied at three dose levels in male rats following a single dermal application of 14C labelled test substance, simulating contact with the formulation concentrate and aqueous spray dilutions thereof. The target dose levels were 500 μg/cm2 [corresponding nominally to about 5000 μg/animal and about 16 mg/kg body weight (calculated for a mean actual body weight of 307 g)], 5.0 μg/cm2 [corresponding nominally to about 50.0 μg/animal and about 0.16 mg/kg body weight (calculated for a mean actual body weight of 318 g)], and 2.5 μg/cm2 [corresponding nominally to about 25.0 μg/animal and about 0.07 mg/kg body weight (for a mean actual body weight of 338 g)].

Groups of four animals for each time point were exposed according to the following regimen:

Duration of exposure [h]

8

8

8

Skin wash after [h]

8

8+24

8+120

Skin strip after [h]

8

24

120

Sacrifice time [h]

8

24

120

Number of animals

4

4

4

Mean recoveries of radioactivity from all dose groups were in the range of 92.96 to 99.30 % of the total radioactivity applied. The largest proportion of radioactivity was recovered from the skin washes for all dose groups.

For the high-dose, i.e. the formulation concentrate, a mean absorption of 0.37 % of dose was observed after an 8 h exposure period to 14C labelled test substance. At sacrifice after 24 and 120 h, mean absorptions were 0.69 and 1.22 % of dose, respectively. In the high-dose group, less than 30 % of the radioactivity remaining in the skin after the end of exposure penetrated through the skin during the 5-day post-observation period.

For the mid-dose, i.e. a 1:100 (v:v) aqueous dilution of the formulation concentrate, a mean absorption of 2.89 % was observed immediately after the 8 h exposure period to 14C labelled test substance. At sacrifice after 24 and 120 h, mean absorptions were 1.87 and 3.48 % of dose, respectively. In the mid-dose group, less than 15 % of the radioactivity remaining in the skin after the end of exposure penetrated through the skin during the 5-day post-observation period.

For the low-dose, i.e. a 1:200 (v:v) aqueous dilution of the formulation concentrate, a mean absorption of 4.25 % was observed immediately after an 8 h exposure period for 14C labelled test substance. At sacrifice after 24 and 120 h, mean absorptions were 4.67 and 5.55 % of the radioactivity applied, respectively. In the low-dose group, less than 15 % of the radioactivity remaining in the skin after the end of exposure penetrated through the skin during the 5-day post-observation period.

The mean percentages of absorbed dose are summarized in the table below: 

mean percentage and ± SD of                    14C labelled test substance
radioactivity absorbed

 

target dose [μg/cm²]

exposure time [h]

sacrifice time [h]

500

5.0

2.5

8

8

0.37±0.11

2.89±2.19

4.25±1.65

8

24

0.69±0.45

1.87±0.68

4.67±0.60

8

120

1.22±0.31

3.48±1.09

5.55±1.08

Overall, low amounts of test substance were absorbed with an increase of relative absorption with decreasing dose of the test substance. Tissue concentrations of radioactivity were generally found at low levels (< 0.1 μg Eq/g), with highest levels found in the blood cells or carcass.

Supporting study: Metabolism 2013/8001821

Following a single oral administration of ME5343-T7 to a rat at a nominal dose of 300 mg/kg bw, the urine and fecal extracts obtained from the animal were analyzed by HPLC-QTOF-MS to detect ME5343-T7 and its possible metabolites, such as ME5343, ME5343-Tl, ME5343-T2 and ME5343-T3. For comparative purpose, the control samples from a rat given the vehicle only were also prepared. No detectable peaks were noted for any samples from the untreated control animal. ME5343-T7 and a trace of ME5343 were detected in the fecal extracts from the treated animal, but no other metabolites were detected. In the urine sample of the treated animal, no detectable test substance/metabolites were noted. The results indicate that ME5343-T7 is excreted only though fecal route without intensive biotransformation to ME5343-Tl, ME5343-T2 and ME5343-T3, and the absorption rate or bioavailability is assumed to be very low, although contribution of the biliary excretion cannot be excluded to make a final conclusion. When the dose formulation was analyzed by the same manner, ME5343 was also detected at a notable level indicating it was present as an impurity in the dose. The area count ratio of ME5343 toward ME5343-T7 was calculated to be 2.6 x 10-3 and 8.0 x 10-3 for the fecal sample and the dose formulation, respectively The result indicates the ratio of ME5343 is higher in the dose formulation than that in the fecal extracts, and no significant production of ME5343 was observed in the biological sample beyond the level of the impurity. The results obtained here showed no positive evidence that ME5343-T7 biotransforms to ME5343 or to typical metabolites, ME5343-Tl, ME5343-T2 and ME5343-T3, formed by the rat in the metabolism of ME5343.

Supporting study: Metabolism 2012/8000521

A preliminary metabolism study of [NCA-14C] labelled test substance in rats was performed in order to make an appropriate study plan for a subsequent definitive metabolism study with the test substance. For this purpose, [NCA-14C] labeled test substance was administrated to male and female rats at a nominal dose of 3 or 300 mg/kg bw, one animal for each experimental group, by a single oral gavage, and excretion rate, mass balance and tissue distribution at termination were determined. In addition, profiles of metabolites in urine and feces were also investigated.

Following a single oral administration of [NCA-14C] labelled test substance to rats, the dosed radioactivity was readily excreted through the urine and feces. The results indicated that fecal excretion was the predominant route for elimination of the test substance in rats, irrespective of the dose level and sex. No significant radioactivity was found in the expired air. Radioactivity remaining in the body (Carcass + GI tract) at 96 h post dose was 0.48 to 1.03 % AD. Recovery of administered dose was 94-95 %.

96 hr post dose

Percentage of the administered dose (%AD)

3 mg/kg bw

300 mg/kg bw

Male

Female

Male

Female

Expired air

0.06

0.08

0.05

0.06

Urine

6.75

5.73

13.60

15.65

Feces

87.86

87.37

81.14

78.71

Cage wash

0.24

0.07

0.22

0.16

Total excreted

94.92

93.25

95.01

94.58

GI tract and contents

0.06

0.61

0.05

0.06

Carcass

0.52

0.42

0.43

0.41

Total remaining in the body

0.57

1.03

0.48

0.48

Total

95.49

94.28

95.49

95.06

Tissue distribution of the radioactivity was investigated at 96 hour post dose. At the low dose level, the highest residue level was found in liver (0.028 to 0.042 mg eq./kg) followed by adrenals, heart muscle and kidneys (0.025 to 0.038 mg eq./kg), and all other tissues were no greater than 0.02 mg eq./kg. At the high dose level, the highest residue level was found in liver (2.15 to 3.46 to mg eq./kg), followed by heart muscle, adrenals and kidneys (2.10 to 2.89 mg eq./kg), and all other tissues were no greater than 2 mg eq./kg. 14C-concentrations in the plasma were 0.0003 to 0.0004 mg eq./L and 0.03 to 0.04 mg eq./L for the low and high dose levels, respectively, and were much lower than those in the other tissues.

The metabolites detected at >5 %AD in either urine or feces were ME5343-Tl, ME5343-T2, ME5343-T8 and an unknown metabolite "HPLC Reg.#25". Minor metabolites, such as ME5343-T9 and other unknowns were also detected at the level lower than 5 %AD. Significant amount (28 to 39 %AD) of unchanged ME5343 was found only in feces. Profiles of metabolites observed in urine were relatively similar with those in feces except for presence of unchanged ME5343 in the feces.

The main metabolic reactions of the test substance in rats, based on the preliminary study, are considered to be 1) hydrolytic elimination of both 3- and 4-positions of cyclopropanecarbonyl groups, and 2) hydroxylation at 4-methyl group. The primary reaction likely to be hydrolysis of 3-cyclopropanecarbonyl group forming ME5343-T2, then further hydrolysis or hydroxylation proceed to produce ME5343-Tl and ME5343-T8, and combination of both reactions leads to form ME5343-T9. No significant sex related differences were observed in excretion route and metabolic fate of [NCA-14C] labelled test substance in rats.