Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 2019 - February 2020

Reliability:

1 (reliable without restriction)

Objective of study:

toxicokinetics

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

F344/DuCrlCrlj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc. (Shiga)
- Age at study initiation: 6 weeks
- Weight at study initiation: 186-206 g (males), 119-138 g (females)
- Housing: 5 animals of the same sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 16 days (males), 14 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): more than 10 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: June 13, 2019 To: July 24, 2019

Route of administration:

oral: feed

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

12.5 mg/kg bw/day (nominal)

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

6 anmials per sex per dose

Control animals:

no

Details on dosing and sampling:

BAS 440 I (Afidopyropen) was offered to six groups of 6 male and 6 female rats in the diet at dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Achieved concentrations, stability and homogeneity were verified by analysis. The animals were observed each day for any evident signs of toxicity. The body weights of all animals were determined once a week and food consumption was determined weekly. Blood samples were taken from all animals before the treatment, and at 21 and 28 days of treatment. At the end of the treatment period, all animals were sacrificed.

Table 1: Maximum plasma concentrations and internal doses (given as AUD) of male and female F344 rats at 21 and 28 days of administration of BAS 440 I
(Afidopyropen)

Compound	Target dose (mg/kg/day)	Males				Females
		Cmax (ng/mL)		AUD (ng*h/mL)		Cmax (ng/mL)		AUD (ng*h/mL)
		D21	D28	D21	D28	D21	D28	D21	D28
BAS 440 I (Afidopyropen)	0.1	0.411	0.445	6.05	6.85	0.315	0.285	4.89	3.82
	0.5	2.23	1.73	31.1	30.1	1.18	1.06	18.6	18.7
	2.5	13.3	12.6	227	221	7.78	7.56	137	112
	12.5	91.1	81	1273	1290	69.5	69.9	1134	1011
	50	n/c	448	n/c	5770	514	583	7860	8598
	150	n/c	855	n/c	8625	n/c	707	n/c	12947
M440I017 (Reg No. 6045738)	0.1	0.157	0.174	3.09	3.18	0.14	0.204	2.77	3.03
	0.5	0.676	0.668	11.5	11.1	0.511	0.566	8.12	9.62
	2.5	4.01	3.6	61	56.9	3.14	2.94	53	46.9
	12.5	18.2	16	330	287	19.5	17.1	331	297
	50	42.3	37.6	760	731	60.6	51	807	808
	150	148	125	2380	2075	119	81.1	1437	1234
M440I001 (Reg. No. 5741530)	0.1	n/c	n/c	n/c	n/c	n/c	n/c	n/c	n/c
	0.5	0.261	0.233	3.51	3.3	0.228	0.211	4.27	4.03
	2.5	1.36	1.21	21.5	18.7	1.1	1.35	20.9	20.2
	12.5	2.77	8.26	149	121	6.7	6.92	124	119
	50	48.5	45.4	723	579	67.3	57.5	925	828
	150	378	316	4675	3323	372	257	5405	3883
M440I060 (Reg. No. 6009307)	0.1	n/c	n/c	n/c	n/c	n/c	n/c	n/c	n/c
	0.5	34.1	34.5	710	716	n/c	28.8	n/c	633
	2.5	146	164	2970	3167	109	130	2000	2453
	12.5	828	939	16267	18450	647	752	12517	13817
	50	3698	4780	67600	82483	5072	5558	82567	89750
	150	10372	13038	194167	214833	5140	8930	87483	149833

n/c: could not be calculated

The maximum plasma concentration increased with increasing dosage of BAS 440 I (Afidopyropen) and its metabolites M440I017 (BASF Reg. No. 6045738), M440I001 (BASF Reg. No. 5741530), and M440I060 (BASF Reg. No. 6009307) for both sampling intervals of study days 21 and 28 (except for the groups where values were out of the quantitation range). The mean maximum plasma concentrations were assessed to be generally comparable (especially when experimental variability was taken into consideration) for the dose groups when compared between the sampling intervals at study day 21 and study day 28. These data demonstrate that under the chosen experimental conditions, kinetics was under a steady state at these sampling time points.

mean maximum plasma concentration of BAS 440 I (Afidopyropen) was also assessed to be linear to sublinear between the low doses tested (with factors between the mean maximum plasma concentrations of 5.4 and 3.9 as well as 3.7 and 3.7 for an increase of the target dose from 0.1 to 0.5 mg/kg/day and the sampling time intervals at study days 21 and 28 as well as for male and female animals, respectively). However, when the target dose was increased from 0.5 to 2.5 and from 2.5 to 12.5 mg/kg/day (reflecting dose factors of 5 and 5), the mean maximum plasma concentrations of BAS 440 I (Afidopyropen) increased by factors of 6.0 and 6.8 and 7.3 and 6.4 as well as 6.6 and 8.9 and 7.1 and 9.2 for sampling intervals at study days 21 and 28 and for males as well as for female animals, respectively. By increasing the target dose further from 12.5 to 50 mg/kg/day (dose factor of 4), the mean maximum plasma concentration of BAS 440 I (Afidopyropen) in male rats increased by a factor of 5.5 at sampling day 28 (mean plasma concentrations at target doses of 50 and 150 mg/kg/day at sampling day 21 could not be calculated). Further increase of the dose to 150 mg/kg/day did result in a 1.9-fold increase in mean maximum plasma concentration at sampling day 28. It is hypothesized that this under proportional increase for BAS 440 I (Afidopyropen) at the high dose is an artifact based on an underestimated mean maximum plasma concentration as plasma concentrations were above the upper limit of quantitation. When the target dose was increased from 12.5 to 50 mg/kg/day (dose factor of 4), the mean maximum plasma concentration of BAS 440 I (Afidopyropen) in female rats increased by factors of 7.4 and 8.3
at sampling days 21 and 28. Further increase of the dose to 150 mg/kg/day in female rats did result in a 1.2-fold increase in mean maximum plasma concentration at the sampling intervals of study day 28. This under proportional increase for BAS 440 I (Afidopyropen) at the high dose may be an artifact based on an underestimated mean maximum plasma concentration as plasma  concentrations were above the upper limit of quantitation.
The dependency of the mean maximum plasma concentrations of metabolite M440I001 (BASF Reg. No. 5741530) showed the same general trend as observed for BAS 440 I (Afidopyropen). When the target dose was increased from 0.5 to 2.5, from 2.5 to 12.5, from 12.5 to 50, and from 50 to 150 mg/kg/day (reflecting dose factors of 5, 5, 4, and 5), the mean maximum plasma concentrations of M440I001 in male rats increased by factors of 5.2, 6.4, 5.5, and 7.8, respectively. In female rats, the mean maximum plasma concentrations of M440I001 increased by factors of 5.2, 6.1, 10.0, and 5.5 when the target dose of BAS 440 I (Afidopyropen) was increased from 0.5 to 2.5, from 2.5 to 12.5, from 12.5 to 50, and from 50 to 150 mg/kg/day. The expected linear increase of the mean
maximum plasma concentrations between the low doses could not be assessed for this metabolite, since for the low dose level of 0.1 mg/kg/day, mean maximum plasma concentrations were not available (based on values below the limit of quantitation).
These dose dependent data for BAS 440 I (Afidopyropen) and its metabolite M440I001 (BASF Reg. No. 5741530) demonstrated, that mean maximum plasma concentrations increased linear to sublinear between the lowest doses tested (data for BAS 440 I) whereas with increasing dose level (from 0.5 mg/kg/day to the next higher doses), the mean maximum plasma concentrations were increasing in a disproportional (supralinear) manner (data for BAS 440 I and its metabolite
M440I001). These data demonstrated that plasma kinetics after dosing BAS 440 I (Afidopyropen) was linear at low doses but started to be saturated at dose levels over 0.5 mg/kg/day under the test conditions used.
For the metabolites M440I017 (BASF Reg. No. 6045738) and M440I060 (BASF Reg. No. 6009307), a more or less sublinear to linear increase of the mean maximum plasma concentrations was observed.

Table 2: Factors of external doses (mean actual doses) and internal doses (given as AUD) of male F344 rats at 21 days of administration of BAS 440 I (Afidopyropen)

Compound	Target dose (mg/kg/day)	Mean actual dose (mg/kg/day)	AUD (ng*h/mL)	Dose factor related to low dose	Dose factor related to previous dose	AUD factor related to low dose	AUD factor related to previous dose
BAS 440 I (Afidopyropen)	0.1	0.1	6.05	NA	NA	NA	NA
	0.5	0.49	31.1	4.9	4.9	5.1	5.1
	2.5	2.49	227	24.9	5.1	37.5	7.3
	12.5	12.6	1273	126	5.1	210.4	5.6
	50	50.3	n/c	503	4	n/c	n/c
	150	154	n/c	1540	3.1	n/c	n/c
M440I017 (Reg. No. 6045738)	0.1	0.1	3.09	NA	NA	NA	NA
	0.5	0.49	11.5	4.9	4.9	3.7	3.7
	2.5	2.49	61	24.9	5.1	19.7	5.3
	12.5	12.6	330	126	5.1	106.8	5.4
	50	50.3	760	503	4	246	2.3
	150	154	2380	1540	3.1	770.2	3.1
M440I001 (Reg. No. 5741530)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.49	3.51	NA	4.9	n/c	n/c
	2.5	2.49	21.5	5.1	5.1	6.1	6.1
	12.5	12.6	149	25.7	5.1	42.5	6.9
	50	50.3	723	102.7	4	206	4.9
	150	154	4675	314.3	3.1	1331.9	6.5
M440I060 (Reg. No. 6009307)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.49	710	NA	4.9	n/c	n/c
	2.5	2.49	2970	5.1	5.1	4.2	4.2
	12.5	12.6	16267	25.7	5.1	22.9	5.5
	50	50.3	67600	102.7	4	95.2	4.2
	150	154	194167	314.3	3.1	273.5	2.9

NA: Not applicable
n/c: could not be calculated.
Dose factor calculated for mean actual nominal dose.
Dose factor calculated relative to lowest dose with calculated AUD value.

Table 3: Factors of external doses (mean actual doses) and internal doses (given as AUD) of male F344 rats at 28 days of administration of BAS 440 I (Afidopyropen)

Compound	Target dose (mg/kg/day)	Mean actual dose (mg/kg/day)	AUD (ng*h/mL)	Dose factor related to low dose	Dose factor related to previous dose	AUD factor related to low dose	AUD factor related to previous dose
BAS 440 I (Afidopyropen)	0.1	0.1	6.85	NA	NA	NA	NA
	0.5	0.49	30.1	4.9	4.9	4.4	4.4
	2.5	2.49	221	24.9	5.1	32.3	7.3
	12.5	12.6	1290	126	5.1	188.3	5.8
	50	50.3	5770	503	4	842.3	4.5
	150	154	8625	1540	3.1	1259.1	1.5
M440I017 (Reg. No. 6045738)	0.1	0.1	3.18	NA	NA	NA	NA
	0.5	0.49	11.1	4.9	4.9	4.9	3.5
	2.5	2.49	56.9	24.9	5.1	17.9	5.1
	12.5	12.6	287	126	5.1	90.3	5
	50	50.3	731	503	4	229.9	2.5
	150	154	2075	1540	3.1	652.5	2.8
M440I001 (Reg. No. 5741530)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.49	3.3	NA	4.9	n/c	n/c
	2.5	2.49	18.7	5.1	5.1	5.7	5.7
	12.5	12.6	121	25.7	5.1	36.7	6.5
	50	50.3	579	102.7	4	175.5	4.8
	150	154	3323	314.3	3.1	1007	5.7
M440I060 (Reg. No. 6009307)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.49	716	NA	4.9	n/c	n/c
	2.5	2.49	3167	5.1	5.1	4.4	4.4
	12.5	12.6	18450	25.7	5.1	25.8	5.8
	50	50.3	82483	102.7	4	115.2	4.5
	150	154	214833	314.3	3.1	300	2.6

NA: Not applicable
n/c: could not be calculated.
Dose factor calculated for mean actual nominal dose.
Dose factor calculated relative to lowest dose with calculated AUD value.

Table 3: Factors of external doses (mean actual doses) and internal doses (given as AUD) of female F344 rats at 21 days of administration of BAS 440 I (Afidopyropen)

Compound	Target dose (mg/kg/day)	Mean actual dose (mg/kg/day)	AUD (ng*h/mL)	Dose factor related to low dose	Dose factor related to previous dose	AUD factor related to low dose	AUD factor related to previous dose
BAS 440 I (Afidopyropen)	0.1	0.1	4.89	NA	NA	NA	NA
	0.5	0.5	18.6	5	5	3.8	3.8
	2.5	2.46	137	24.6	4.9	28	7.4
	12.5	12.5	1134	125	5.1	231.9	8.3
	50	48.2	7860	482	3.9	1607.4	6.9
	150	139	n/c	1390	2.9	n/c	n/c
M440I017 (Reg. No. 6045738)	0.1	0.1	2.77	NA	NA	NA	NA
	0.5	0.5	8.12	5	5	2.9	2.9
	2.5	2.46	53	24.6	4.9	19.1	6.5
	12.5	12.5	331	125	5.1	119.5	6.2
	50	48.2	807	482	3.9	291.3	2.4
	150	139	1437	1390	2.9	518.8	1.8
M440I001 (Reg. No. 5741530)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.5	4.27	NA	5	n/c	n/c
	2.5	2.46	20.9	4.9	4.9	4.9	4.9
	12.5	12.5	124	25	5.1	29	5.9
	50	48.2	925	96.4	3.9	216.6	7.5
	150	139	5405	278	2.9	1265.8	5.8
M440I060 (Reg. No. 6009307)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.5	n/c	NA	5	n/c	n/c
	2.5	2.46	2000	NA	4.9	n/c	n/c
	12.5	12.5	12517	5.1	5.1	6.3	6.3
	50	48.2	82567	19.6	3.9	41.3	6.6
	150	139	87483	56.5	2.9	43.7	1.1

NA: Not applicable
n/c: could not be calculated.
Dose factor calculated for mean actual nominal dose.
Dose factor calculated relative to lowest dose with calculated AUD value.

Table 4: Factors of external doses (mean actual doses) and internal doses (given as AUD) of female F344 rats at 28 days of administration of BAS 440 I (Afidopyropen)

Compound	Target dose (mg/kg/day)	Mean actual dose (mg/kg/day)	AUD (ng*h/mL)	Dose factor related to low dose	Dose factor related to previous dose	AUD factor related to low dose	AUD factor related to previous dose
BAS 440 I (Afidopyropen)	0.1	0.1	3.82	NA	NA	NA	NA
	0.5	0.5	18.7	5	5	4.9	4.9
	2.5	2.46	112	24.6	4.9	29.3	6
	12.5	12.5	1011	125	5.1	264.7	9
	50	48.2	8598	482	3.9	2250.8	8.5
	150	139	12947	1390	2.9	3389.3	1.5
M440I017 (Reg. No. 6045738)	0.1	0.1	3.03	NA	NA	NA	NA
	0.5	0.5	9.62	5	5	3.2	3.2
	2.5	2.46	46.9	24.6	4.9	15.5	4.9
	12.5	12.5	297	125	5.1	98	6.3
	50	48.2	808	482	3.9	266.7	2.7
	150	139	1234	1390	2.9	407.3	1.5
M440I001 (Reg. No. 5741530)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.5	4.03	NA	5	n/c	n/c
	2.5	2.46	20.2	4.9	4.9	5	5
	12.5	12.5	119	25	5.1	29.5	5.9
	50	48.2	828	96.4	3.9	205.5	7
	150	139	3883	278	2.9	963.5	4.7
M440I060 (Reg. No. 6009307)	0.1	0.1	n/c	NA	NA	NA	NA
	0.5	0.5	633	NA	5	n/c	n/c
	2.5	2.46	2453	4.9	4.9	3.9	3.9
	12.5	12.5	13817	25	5.1	21.8	5.6
	50	48.2	89750	96.4	3.9	141.8	6.5
	150	139	149833	278	2.9	236.7	1.7

NA: Not applicable
n/c: could not be calculated.
Dose factor calculated for mean actual nominal dose.
Dose factor calculated relative to lowest dose with calculated AUD value.

 

The mean AUDs of BAS 440 I (Afidopyropen) in male rats at the sampling interval of study day 21 were 6.05, 31.1, 227, and 1273 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, and 12.5 mg/kg/day. For the dose levels of 50 and 150 mg/kg/day, the AUD could not be calculated.
Mean AUDs of BAS 440 I (Afidopyropen) in male rats at the sampling interval of study day 28 were 6.85, 30.1, 221, 1290, 5770, and 8625 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of BAS 440 I (Afidopyropen) in female rats at the sampling interval of study day 21 were 4.89, 18.6, 137, 1134, and 7860 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, 12.5, and 50 mg/kg/day. For the dose level of 150 mg/kg/day, the AUD could not be calculated.
Mean AUDs of BAS 440 I (Afidopyropen) in female rats at the sampling interval of study day 28 were 3.82, 18.7, 112, 1011, 8598, and 12947 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day.
Mean AUDs of metabolite M440I017 (BASF Reg. No. 6045738) in male rats at the sampling interval of study day 21 were 3.09, 11.5, 61, 330, 760, and 2380 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of metabolite M440I017 in male rats at the sampling interval of study day 28 were 3.18, 11.1, 56.9, 287, 731, and 2075 ng*h/mL for the target
dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of metabolite M440I017 in female rats at the sampling interval of study day 21 were 2.77, 8.12, 53, 331, 807, and 1437 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of metabolite M440I017 in female rats at the sampling interval of study day 28 were 3.03, 9.62, 46.9, 297, 808,
and 1234 ng*h/mL for the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day.
Mean AUDs of metabolite M440I001 (BASF Reg. No. 5741530) in male rats at the sampling interval of study day 21 were 3.51, 21.5, 149, 723, and 4675 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of metabolite M440I001 in male rats at the sampling interval of study day 28 were 3.3, 18.7, 121, 579, and 3323 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. For both sampling intervals at the target dose level of 0.1 mg/kg/day, the AUDs could not be calculated since measured plasma concentrations were below the limit of quantitation. Mean AUDs of metabolite M440I001 in female rats at the sampling interval of study day 21 were 4.27, 20.9, 124, 925, and 5405 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of metabolite M440I001 in female rats at the sampling interval of study day 28 were 4.03, 20.2, 119, 828, and 3883 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Corresponding to the situation in male rats, for both sampling intervals at the target dose level of 0.1 mg/kg/day, the AUDs could not be calculated since measured plasma concentrations were below the limit of quantitation. Mean AUDs of metabolite M440I060 (BASF Reg. No. 6009307) in male rats at the sampling interval
of study day 21 were 710, 2970, 16267, 67600, and 194167 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. Mean AUDs of metabolite M440I060 in male rats at the sampling interval of study day 28 were 716, 3167, 18450, 82483, and 214833 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. For both sampling intervals at the target dose
level of 0.1 mg/kg/day, the AUDs could not be calculated since measured plasma concentrations were below the limit of quantitation. Mean AUDs of metabolite M440I060 in female rats at the sampling interval of study day 21 were 2000, 12517, 82567, and 87483 ng*h/mL for the target dose levels of 2.5, 12.5, 50, and 150 mg/kg/day. For the target dose levels of 0.1 and 0.5 mg/kg/day, the AUD could not be calculated since measured plasma concentrations were below the limit of quantitation. Mean AUDs of metabolite M440I060 in female rats at the sampling interval of study day 28 were 633, 2453, 13817, 89750, and 149833 ng*h/mL for the target dose levels of 0.5, 2.5, 12.5, 50, and 150 mg/kg/day. For the target dose level of 0.1 mg/kg/day, the AUD could not be calculated since measured plasma concentrations were below the limit of quantitation.
As can be seen from the mean AUDs of BAS 440 I (Afidopyropen) and its metabolites M440I017 (BASF Reg. No. 6045738), M440I001 (BASF Reg. No. 5741530), and M440I060 (BASF Reg. No. 6009307), the internal doses (given as AUDs) raised with increasing dosage for both sampling intervals of study days 21 and 28 (except for the groups where no values could be calculated). The mean AUDs for all analytes were assessed to be generally comparable for the dose groups for both sexes, when compared between the sampling intervals at study day 21 and study day 28. These data on internal doses demonstrate that under the chosen experimental conditions, kinetics was in a steady state situation at these sampling time points. Therewith, this finding, based on internal doses, paralleled the conclusion of steady state as derived from the assessments of the mean maximum plasma concentrations.

A more or less sublinear to linear increase of the mean AUDs over the external (mean actual dose) was observed for the metabolite M440I017 (BASF Reg. No. 6045738). For the metabolite M440I060 (BASF Reg. No. 6009307), the relationship between the internal and external dose was partly linear, however with several data points, more pronounced at higher doses and more pronounced in female than in male rats, that indicated the tendency of higher factors for internal than for external dose increases, e.g. for female animals at the sampling interval at 21 days, where an increase of the mean actual doses from 2.46 to 12.5 and from 12.5 to 48.2 mg/kg/day (5.1- and 3.9-fold) resulted in an increase in the corresponding AUDs from 2000 to 12517 to 82567 ng*h/mL, reflecting an increase
in internal doses of factors of 6.3 and 6.6. For BAS 440 I (Afidopyropen), its mean AUDs were also assessed to be linear to sublinear between the low doses tested with factors between the mean AUDs of 5.1 and 5.1 and 4.4 and 4.4 as well as
3.8 and 3.8 and 4.9 and 4.9 for an increase of the mean actual doses from 0.1 to 0.49 mg/kg/day (males) and 0.1 to 0.5 mg/kg/day (females), the sampling time intervals at study days 21 and 28 as well as for male and female animals. However, when the target dose was increased from 0.5 to 2.5
and from 2.5 to 12.5 mg/kg/day (reflecting factors of the mean actual doses of 5.1 and 5.1 for males as well as 4.9 and 5.1 for females), the corresponding mean AUDs of BAS 440 I (Afidopyropen) increased by factors of 7.3 and 5.6 as well as 7.4 and 8.3 for males and females, respectively for
samples taken on study day 21 and by factors of 7.3 and 5.8 as well as 6.0 and 9.0 for males and females, respectively for samples taken on study day 28.
By increasing the target dose further from 12.5 to 50 mg/kg/day (factor between the actual doses of 4.0), the mean AUDs of BAS 440 I (Afidopyropen) in male rats increased by a factor of 4.5 at sampling day 28 (AUDs at target doses of 50 and 150 mg/kg/day on sampling day 21 could not be calculated). Further increase of the dose to 150 mg/kg/day did result in a 1.5-fold increase in AUD.
It is hypothesized that this under proportional increase for BAS 440 I (Afidopyropen) at the high dose is an artifact based on an underestimated mean maximum plasma concentration at this dose level that is based on plasma samples in this dose group that resulted plasma concentrations of BAS 440 I
(Afidopyropen) were above the upper limit of quantitation. When the target dose was increased from 12.5 to 50 mg/kg/day (factor of actual doses of 3.9), the mean AUDs of BAS 440 I (Afidopyropen) in female rats increased by factors of 6.9 and 8.5 at sampling days 21 and 28. Further increase of the dose to 150 mg/kg/day in female rats did result in a 1.5-fold increase in mean AUD at the sampling intervals of study day 28. As already outlined for males, it is also hypothesized here that this under proportional increase for BAS 440 I (Afidopyropen) at the high dose is an artifact based on an underestimated mean maximum plasma concentration at this dose level that is based on plasma
samples in this dose group that resulted plasma concentrations of BAS 440 I (Afidopyropen) were above the upper limit of quantitation. Data on sampling day 21 could not be calculated.
The dependency of the mean AUDs of metabolite M440I001 (BASF Reg. No. 5741530) showed a general trend as observed for BAS 440 I (Afidopyropen). When the target dose was increased from 0.5 to 2.5, from 2.5 to 12.5, from 12.5 to 50, and from 50 to 150 mg/kg/day (reflecting dose factors of 5.1, 5.1, 4.0, and 3.1 in males as well as 4.9, 5.1, 3.9, and 2.9 in females), the mean AUDs of
M440I001 in male rats increased by factors of 6.1, 6.9, 4.9, and 6.5 (sampling on study day 21) and 5.7, 6.5, 4.8, and 5.7 (sampling on study day 28). In female rats, the corresponding mean AUDs of M440I001 increased by factors of 4.9, 5.9, 7.5, and 5.8 (sampling on study day 21) and 5.0, 5.9, 7.0, and 4.7 (sampling on study day 28). The expected linear increase of the mean AUDs between the
lowest test doses could not be assessed for this metabolite, since for the low dose level of 0.1 mg/kg/day, mean AUDs were not available. However, for female animals, the internal dose of M440I001 was still linear to the external (actual dose) for female animals between the actual doses of 0.5 and 2.46 mg/kg/day.These dose dependent data for BAS 440 I (Afidopyropen) and its metabolite M440I001 (BASF Reg. No. 5741530) demonstrate that mean AUDs increased linear to sublinear between the lowest doses tested (between 0.1 and 0.5 mg/kg/day) in BAS 440 I (Afidopyropen) and in M440I001 for doses
between 0.5 to 2.5 mg/kg/day in female animals. Since low dose groups of 0.1 mg/kg/day for both sexes and for both target analytes resulted in values of plasma concentrations below the limit of quantitation, for the calculation of pharmacokinetic parameters, these values were set to the lower limit of quantitation, resulting therewith in a potential overestimation of the calculated internal doses.
Consequently, the given factors for the AUDs between these doses should be assessed carefully within the context of these procedures and within their given variability. For higher doses, the mean AUDs were increasing in a disproportional (supralinear) manner (data for BAS 440 I (Afidopyropen) and its metabolite M440I001). These data demonstrated that plasma kinetics after dosing BAS 440 I
(Afidopyropen) were linear at low doses but start to be saturated at higher dose levels under the test conditions used. Whereas further increase of the external dose from 50 to 150 mg/kg/day (factor 3 of actual doses of 3.1 for males and 2.9 for females) resulted in a sublinear increase of the mean AUDs of BAS 440 I (Afidopyropen) for the sampling on study day 28 that were postulated to be an
artifact based on analytical results for BAS 440 I (Afidopyropen) in many plasma samples resulted in values above the upper limit of quantitation. The mean AUDs of its metabolite M440I001 further increased supralinear for both sampling days and for both sexes providing further evidence of saturation of kinetics at higher doses for the scenario tested.

Conclusions:

The objective of this study was to evaluate the pharmacokinetics of BAS 440 I (Afidopyropen) and of its three metabolites M440I017 (BASF Reg. No. 6045738), M440I001 (BASF Reg. No. 5741530), and M440I060 (BASF Reg. No. 6009307) in rats. F344 rats (F344/DuCrlCrlj) of both sexes were fed diets containing the test substance at concentrations adjusted to equate the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day every week for a period of 28 days (4 weeks). Each dose group consisted of 6 animals per sex. Pharmacokinetic analyses were conducted for both sexes based on blood samplings at days 21 and 28 and the concentrations of BAS 440 I (Afidopyropen) and the three metabolites M440I017 (BASF Reg. No. 6045738), M440I001 (BASF Reg. No. 5741530), and M440I060 (BASF Reg. No. 6009307) were quantified in each individual plasma sample. Under the chosen test conditions, it could be demonstrated in principle that BAS 440 I (Afidopyropen) and the metabolites M440I001 and M440I060 show a linear kinetics at low doses tested but switch to a disproportionate increase in internal doses over external doses when the external dose level exceeds 0.5 mg/kg/day.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From: 2015-06-08 To: 2016-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Tests on In Vivo Fate in Animals

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

NON RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 080722.
- Purity: 98.50 %
- Appearance: Yellow solid

STORAGE CONDITIONS OF TEST MATERIAL
- at room temperature

CPCA-carnitine (M440I060, 15/0440-1)
- Lot/batch No.of test material: L82-166.
- Purity: 97.80 %

STORAGE CONDITIONS OF TEST MATERIAL
- in refrigerator (2-8°C)

Reg.No. 6045738 (M440I017, 15/0199-2)
- Lot/batch No.of test material: L82-178.
- Purity: 94.80 %

STORAGE CONDITIONS OF TEST MATERIAL
- in refrigerator (2-8 °C)

Reg.No. Reg.No. 5741530 (M440I001, AS1561)
- Lot/batch No.of test material: L82-66.

STORAGE CONDITIONS OF TEST MATERIAL
- in refrigerator (2-8°C)

RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
solution in acetonitrile
- Lot/batch No.of test material: Batch 1055-0201.

RADIOLABELLING INFORMATION
- Purity: 95.60 %
- Radiochemical purity: 99.00 %
- Specific activity 3.7 MBq/mg; concentration in acetonitrile 4.47 mg/g (16.7 MBq/g solution)
- Locations of the label: nicotinic acid 9-14CShort

STORAGE CONDITIONS OF TEST MATERIAL
- in freezer (≤ -15 °C)

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344/DuCrj

Remarks:

F344 Rat (Japanese clone) (F344/DuCrlCrj)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan.
- Age at study initiation: 13-16 weeks
- Housing: individually housed
- Diet: ground Kliba rat/mouse maintenance diet “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: At least 14 days under laboratory conditions. Animals were dosed orally at least twice during the acclimatization period with milli-Q in order to train the animals.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-21 °C
- Humidity: 32-78 %
- Air changes: at least 10 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2015-06-26 To: 2015-07-23

Route of administration:

other: oral: gavage and oral: feed

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The non-radioactive dietary mixtures administered for the first 14 days were mixed once using powdered rodent diet (ground Kilba rat/mouse, Switzerland) and a premix. For the radioactive oral doses the acetonitrile was evaporated to dryness and then stock solutions were prepared in 0.5% carboxymethylcellulose in tap water.

Duration and frequency of treatment / exposure:

15 days (14 days oral:feed + 1 day oral: gavage)

Remarks:

36 ppm (Day 1 till Day 14) + 3 mg/kg bw (Day 15)

Remarks:

180 ppm (Day 1 till Fay 14) + 15 mg/kg bw Day 15)

Remarks:

600 ppm (Day 1 till Day 14) + 50 mg/kg bw (Day 15)

No. of animals per sex per dose / concentration:

4 females per dose

Control animals:

no

Positive control reference chemical:

no

Details on study design:

The study investigated the toxicokinetic parameters of adsorption, distribution, metabolism and excretion of the test compound in the F344 rat after a 14-day dietary administration of the test compound followed by an oral dose of 14C labelled test compound on Day 15.

Details on dosing and sampling:

After administration of the radio-active dose animals were placed in macrolon cages and blood samples (300 μL) were taken from the tail vein into K2-EDTA anticoagulant. Blood, urine and faeces were collected form groups 1, 2 and 3. Groups 4 and 5 were sacrificed after dose administration and the liver and uterus were collected.

Plasma was separated from blood by centrifugation and radioactivity in plasma and urine was determined by solubilization/LSC. Faeces were homogenized and combusted prior to LSC. The liver and uterus were homogenized and solubilized using Hionoc Fluor as the scintillation fluid.

Quantification of the test compound and three non-radiolabeled metabolites was conducted via LCMS to determine the PK properties of each of these molecules under the conditions of the study.

Metabolic profiling (not identification) was also conducted.

Experimental groups for each dose level (see Table 1)

Kinetic calculations
Concentrations of radioactivity in blood and plasma were calculated as ng equivalents of test compound/g of sample, based on the specific activity of the radiolabelled test compound ([14C] labelled test compound) formulation. For each group, all toxicokinetic parameters were calculated from the curves constructed from individual animals using validated Phoenix WinNonlin 6.3. Non-compartmental analysis was applied. In addition the toxicokinetic parameters were calculated from the curves obtained from the bioanalytical results, the test compound and 3 metabolites (constructed from the average values at each time point) using validated WinNonlin 6.3. Non-compartmental analysis was applied.

Parameters that were calculated see Table 2.

Calculations
All calculations were performed with the complete numerical data. The numerical data were generally shown as rounded values (smaller degree of precision) to increase readability.

All results expressed in weight or concentration units are calculated with the assumption that the parent compound or metabolites have the same molecular mass. Since background radioactivity values of the particular matrices were not subtracted, radioactivity balances, mainly in the case of low concentrations, may exceed 100 %. The mean and standard deviation were used to characterize the data, where appropriate (i.e. radioactivity measurement, concentration, etc).

Cumulative amounts (expressed as percentage of the dose) excreted in urine and faeces were calculated by summation of the amounts excreted in the individual urine and faeces samples per collection period, respectively.

The following relationships were used for the calculation of results contained in the present report.

Total radioactivity concentration (TRR) in [mg/kg] = total counts per sample [Bq]/sample weight [g] x spec. activity of dosed material [Bq/µg]

Total radioactivity in [% of dose per matrix] = total counts per sample [Bq] x total matrix weight [g] x 100/ sample weight [g] x total dose [Bq]

Relative peak area in HPLC elution profiles [% ROI] = area of particular peak x 100/ total peak area ( sum of all ROIs)

Concentration of metabolite [mg/kg] = relative amount of metabolite [% ROI] x concentration of radioactive residues [mg/kg]/100

Amount of metabolite in % of total radioactive residue [% TRR] = concentration of metabolite [mg/kg] x 100/ total radioactive residue [mg/kg] (TRR)

Details on absorption:

In plasma kinetics, AUC values indicated an internal exposure that is clearly correlated to the dosing regimen of 14C labelled test compound. The AUC values of 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. In time, a distribution towards the red blood cells was noted.

In plasma kinetics of the test compound and its metabolites (bioanalytical data), the AUC values of the test compound increased with increasing dose level in an unproportional manner. From 3 to 15 mg/kg test compound 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.

The kinetic data are presented in the Tables 4 -12.

Details on distribution in tissues:

Analysis of the liver and uterus found the majority of radioactivity in the liver (Table 13).

Individual results are given in the Table 14.

Details on excretion:

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 were 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.

The mean results are given in Table 15.

Concentration, homogeneity and stability of dosing suspensions

The non-radiolabeled diets were 90-110% of the target concentration and confirmed to be homogeneous. The radiochemical purity of of the test substance in the dosing suspensions was 90.6 to 100 %. Homogeneity of the dosing suspensions was confirmed by analysis of triplicate aliquots of each suspension (coefficient of variation ≥5 %).

Mortality and clinical signs

There were no treatment-related clinical signs or mortality.

Body weight

No abnormalities of the body weight were observed during the experiments.

Food Consumption

No test substance related effect was noted on absolute food intake or on relative food consumption data (expressed as gram food/kg bw/d).

Table 3: Test substance intake

Group	Nominal dietary dose [ppm]	Average intake [mg /kg bw/d]
1	36	3
2	600	44
3	180	14
4	600	51
5	180	14

Necropsy

Necropsy was only conducted on groups 4 and 5. There were no treatment related changes observed.

Table 4: Mean radioactivity concentrations in plasma of female rats having received an oral dose of ¹⁴C labelled test substance at 3, 15 or 50 mg/kg bw 

	Plasma (Eq/g plasma)			Blood (Eq/g plasma)
Time (h)	3 mg/kg bw	15 mg/kg bw	50 mg/kg bw	3 mg/kg bw	15 mg/kg bw	50 mg/kg bw
1	262	2803	6469	221	2108	4910
2	113	1942	6541	107	1509	4786
4	38.0	1031	4332	47.0	818	3547
8	18.7	206	1646	29.5	201	1333
24	6.00	49.0	146	17.0	75.7	208
48	<LLOQ	<LLOQ	<LLOQ	13.9	59.1	192
72	<LLOQ	<LLOQ	<LLOQ	12.0	40.7	161

Table 5: Pharmacokinetic parameters determined in female rats having received an oral dose of ¹⁴C labelled test substance at 3, 15 or 50 mg/kg bw

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	262	1	24	8.17	728	800
	15	2800	1	24	5.19*	10400	10800
	50	6980	1-2	24	4.21	41400	42300
blood	3	221	1	72	96.5*	1600	3290*
	15	2110	1	72	55.2*	11700	15500*
	50	5050	1-2	72	NR	43100	NR

* approximation

Table 6: Pharmacokinetics in female F344 rats repeated dose (14D) at 3, 15 or 50 mg/kg bw 

External Dose			Plasma test substance
mg/kg/bw/d	Difference	Cmax ug-Eq/mL		Difference	AUC hr*ug-Eq/mL	Difference
3	1X	262		1X	800	1X
15	5X	2800		10.7X	10800	13.5X
50	16.7X	6980		26.7X	42300	53X

Table: 7: Bioanalytical analysis: pooled plasma concentration of test substance after administration of ¹⁴C labelled test substance at dose levels of 3, 15 and 50 mg/kg bw (oral administration) to female rats 

Time [h]	3 mg/kg bw	15 mg/kg bw	50 mg/kg bw
Predose (Day 1)	< 1	< 1	< 1
Predose (Day 15)	< 1	1.77	3.45
Plasma 1	24.7	1497	3250
Plasma 2	7.87	1085	4752
Plasma 4	1.70	427	2372
Plasma 8	< 1	61.4	791
Plasma 24	< 1	1.90	4.23
Plasma 48	< 1	< 1	< 1
Plasma 72	< 1	< 1	< 1

< 1: below lower limit of quantification (=1 ng/mL)

Table 8: Bioanalytical analysis: pooled plasma concentration of metabolite M440I001 after administration of ¹⁴C labelled test substance at dose levels of 3, 15 and 50 mg/kg bw (oral administration) to female rats 

Time [h]	3 mg/kg bw	15 mg/kg bw	50 mg/kg bw
Predose (Day 1)	< 1	< 1	< 1
Predose (Day 15)	< 1	< 1	1.77
Plasma 1	2.10	153	277
Plasma 2	< 1	111	457
Plasma 4	< 1	57.5	344
Plasma 8	< 1	12.6	95.8
Plasma 24	< 1	1.84	6.10
Plasma 48	< 1	< 1	< 1
Plasma 72	< 1	< 1	< 1

< 1: below lower limit of quantification (=1 ng/mL)

Table 9: Bioanalytical analysis: pooled plasma concentration of metabolite M440I017 after administration of ¹⁴C labelled test substance at dose levels of 3, 15 and 50 mg/kg bw (oral administration) to female rats 

Time [h]	3 mg/kg bw	15 mg/kg bw	50 mg/kg bw
Predose (Day 1)	< 1	< 1	< 1
Predose (Day 15)	< 1	1.77	3.83
Plasma 1	3.91	51.6	107
Plasma 2	3.18	51.6	175
Plasma 4	1.27	47.9	130
Plasma 8	< 1	22.6	75.6
Plasma 24	< 1	1.28	3.53
Plasma 48	< 1	< 1	< 1
Plasma 72	< 1	< 1	< 1

< 1: below lower limit of quantification (=1 ng/mL)

Table 10: Bioanalytical analysis: pooled plasma concentration of metabolite M440I060 (AS1574, CPCA-carnitine) after administration of ¹⁴C labelled test substances at dose levels of 3, 15 and 50 mg/kg bw (oral administration) to female rats 

Time [h]	3 mg/kg bw	15 mg/kg bw	50 mg/kg bw
Predose (Day 1)	1.48	1.12	1.05
Predose (Day 15)	39.3	253	1485
Plasma 1	195	729	1333
Plasma 2	182	1045	1653
Plasma 4	148	1022	2458
Plasma 8	139	1562	4187
Plasma 24	66.4	425	2367
Plasma 48	44.5	260	726
Plasma 72	26.4	208	695

Table 11: Bioanalytical analysis: pharmacokinetic parameters of the test substance and its metabolites in plasma after oral administration of ¹⁴C labelled test substance at dose levels of 3, 15 and 50 mg/kg bw to female rats 

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]
BAS 440 I	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

Table 12: Pharmacokinetics of individual residues in the F344 rat. Repeated dietary dose (14D) at 3, 15 or 50 mg/kg bw 

Analyte	External Dose		Plasma
	mg/kg/bw/d	Difference	Cmax ng-Eq/mL	Difference	AUD Ng*h/mL	Difference
Afidopyropen (BAS 440 I)	3	1X	24.7	1X	104	1X
	15	5X	1500	61X	4530	44X
	50	16.7X	4750	192X	20700	199X
M440I001	3	1X	-	-	72.5	1X
	15	5X	153	-	635	8.8X
	50	16.7X	457	-	2690	37X
M440I017	3	1X	3.91	1X	78.2	1X
	15	5X	51.6	13X	482	6X
	50	16.7X	175	45X	1350	17.3X
CPCA-carnitine M440I060	3	1X	195	1X	4930	1X
	15	5X	1560	8X	36200	7.3X
	50	16.7X	4190	22X	122000	25X

Table 13: Mean tissue concentrations of radioactivity in female rats having received an oral dose of ¹⁴C labelled test substance at 15 or 50 mg/kg bw. 

	Results expressed inμg Eq/g tissue		Results expressed in percentage of dose found in tissue
Dose level	15 mg/kg bw	50 mg/kg bw	15 mg/kg bw	50 mg/kg bw
Time after administration (h)	1	1.5	1	1.5
Blood cells	12.3	8.92	0.623	0.492
Plasma	3.45	8.62	0.25	0.184
Uterus	4.43	12.8	0.069	0.082
Liver	42.2	92.7	7.96	5.35

Table 14: Individual tissue concentrations of radioactivity in female rats having received an oral dose of ¹⁴C labelled test substance at 15 or 50 mg/kg bw. (% of dose found in tissue) 

	15 mg/kg bw samples after 1 hour				50 mg/kg bw sampled after 1.5 hours
animal number	17	18	19	20	13	14	15	16
blood cells	ns	0.603	0.655	0.610	0.449	0.529	0.479	0.513
plasma	ns	0.26	0.25	0.24	0.194	0.204	0.147	0.190
uterus	0.060	0.055	0.066	0.096	0.083	0.069	0.0496	0.127
liver	7.96	7.81	7.64	8.42	5.06	5.41	5.93	5.00

Table 15: Mean excretion of retention of radioactivity in female rats having received an oral dose of ¹⁴C labelled test substance at 3, 15 or 50 mg/kg bw. Results are % of dose administered.

Balance/excretion	3 mg/kg/ bw	15 mg/kg bw	50 mg/kg bw
Urine 8 – 24	0.293	0.322	0.720
Urine 24 – 48	0.387	0.639	0.560
Urine 48 – 72	0.237	0.342	0.360
Subtotal Urine	0.917	1.30	1.64
Faeces 8 – 24	34.3	42.5	43.9
Faeces 24 – 48	45.8	41.9	18.5
Faeces 48 – 72	4.70	5.14	2.65
Subtotal Faeces	84.7	89.5	65.0
Total	85.6	90.8	66.6

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From: 2010-12-22 To: 2015-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

excretion

metabolism

other: mass balance

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF (12-Nousan-No.8147, 2-3-1)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

NON RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TA10224601
- Purity: 98.50 %

RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Institute of Isotopes Co., Ltd. (Hungary), XVII/53

RADIOLABELLING INFORMATION
- Radiochemical purity: 95.41 % by radio-HPLC
- Specific activity: 3.29 MBq/mg (1955 MBq/mmol)
- Locations of the label: nicotinic acid 9-14CShort

STORAGE CONDITIONS OF TEST MATERIAL
- in a refrigerator at ca. 5 °C ( defined range: 1 to 10 °C)

Radiolabelling:

yes

Species:

rat

Strain:

other: F344/DuCrlCrlj, SPF/VAF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc. (Kanagawa, Japan)
- Age at study initiation: 9 weeks
- Housing: For the excretion experiment, the animals of the same sex were housed. For the biliary excretion experiment, the surgically prepared bile duct-cannulated animals were individually housed.
- Diet: MF pellet (lot. 100903A1, 101013A2 and 110114A4, Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum
- Water: tap water ad lbitum
- Acclimation period: 4 - 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1 to 23.3 °C
- Humidity: 47 to 67 %
- Air changes: > 20 times/hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2011-01-17 To: 2012-09-12

Route of administration:

oral: gavage

Vehicle:

other: 3 % hydroxylpropyl cellulose solution (3% HPC, w/w). The vehicle was prepared by dissolving hydroxylpropyl cellulose (Wako pure chemical industries, Ltd., Osaka, Japan) in Otsuka distilled water (Otsuka pharmaceutical factory, Inc., Tokushima, Japan).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate aliquot of the stock solution of radiolabeled test substance was mixed with appropriate amount of the non-radiolabeled test substance (as the stock solution for the low dose or the intact crystal for the high dose). The organic solvent was completely removed under gentle stream of nitrogen, and the dried test substance was resuspended in appropriate volume of 3 % HPC solution. The suspension was kept at ambient temperature under the dark condition with continuous stirring until use. The low and high dose formulations were used within 3 days after preparation.

Duration and frequency of treatment / exposure:

single gavage

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

8 per sex per dose

Control animals:

no

Positive control reference chemical:

no

Details on study design:

The test substance was delivered to the stomach of each rat via a gavage tube with a glass syringe. The amount of dose to be administered to each rat was determined based on the body weight measured immediately prior to the dose administration. The oral route was selected for dose administration to be consistent with the relevant toxicological studies, and also as this is expected to be an important route for human exposure.

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.

Details on dosing and sampling:

Samples collected
Expired air: Expired air was not collected in this study, since the preliminary study demonstrated that no significant radioactivity was excreted in the expired air (< 0.1 %AD in 48 hr).
Tissues
At termination, the rat was anesthetized with ethyl ether and its blood was taken entirely from the inferior vena cava with heparinized syringe to humanely kill the animal. Then the gastro-intestinal tract (including the contents) and the remaining carcass were excised. The collected blood was combined with the residual carcass.
Excretion Experiment
Urine: 6, 12, 24, 48, 72 and 96 hr post dose
Feces: 24, 48, 72 and 96 hr post dose
Bile Excretion Experiment
Bile: 6, 24 and 48 hr post dose
Urine: 6, 24 and 48 hr post dose
Feces: 24 and 48 hr post dose

Description of analytical procedures
Sample Processing for Radioassay
Urine
Urine sample was filtered through a filter paper. Duplicate aliquots (0.1 mL) of the filtered urine sample were radioassayed. Residues on the filter paper were subjected to oxidative combustion prior to radioassay. The radioactivity recovered from the filtered urine and the filter paper were summed to determine the total radioactivity in the urine sample. The filtered urine sample was radioassayed on the day of sampling.

Feces
Feces sample was homogenized with an approximately six-fold volume of chilled water using a Polytron® homogenizer under ice-chilled condition and triplicate aliquots (ca. 50 to 200 mg) of the homogenate were solubilized prior to radioassay.

Bile
Duplicate aliquots (0.05 to 0.1 mL) of the bile sample were radioassayed on the day of sampling.

Cage Wash
Cage wash sample was filtered through a filter paper. The filter was rinsed with methanol/water (1/1, v/v) and the rinsate was combined with the filtered sample. Duplicate aliquots (2 mL) of the filtered cage wash were radioassayed. Residues on the filter paper were subjected to oxidative combustion prior to radioassay. The radioactivity recovered from the filtered solution and the filter paper were summed to determine the total radioactivity in the cage wash sample.

Tissues
The gastro-intestinal tract including contents was entirely solubilized with approximately
90 mL of HCl/2-propanol mixture (1/2, v/v) at approximately 50 °C for 18 hrs, and triplicate aliquots of the digest were radioassayed. The residual carcass was homogenized with 10 to 15 mL of 2-propanol/water mixture (1/1, v/v) with an appropriate amount of dry ice using a blender, and triplicate aliquots (ca. 300 mg) of the homogenate were radioassayed following oxidative combustion.

Metabolite Analysis
Sample processing
Pooled samples were prepared for the filtered urine, fecal homogenate and bile samples to use for metabolite analyses. The following samples were used to prepare the pooled samples:
Urine: 0 to 96 hr post dose
Feces: 0 to 96 hr post dose
Bile: 0 to 48 hr post dose
Proportional portions (10 % by volume for the urine and bile, and 10% by weight for the fecal homogenate) of the samples obtained from individual animals at each time interval were all combined for the same test group.
For the pooled urine and the pooled bile, an aliquot was directly subjected to radio-HPLC analysis.

A portion (ca. 4 g) of the pooled fecal homogenate was extracted with approximately 15 mL of acetonitrile using the Polytron® homogenizer under an ice-chilled condition. The homogenate was sonicated for 5 minutes and then centrifuged at 6000 rpm (approx. 4000 ×g) for 5 minutes at 4 °C to separate the supernatant and the pellet. Similar extraction process was repeated for the pellet once with acetonitrile and additional two times with acetonitrile/water (1/1, v/v). All extracts (supernatant after centrifugation) were combined to obtain an extract fraction. The final pellet was suspended in 2 mL of acetonitrile. Duplicate aliquots of the extract and triplicate aliquots of the pellet suspension were radioassayed. The samples of the pellet suspension were solubilized prior to radioassay. Above extractions were performed in duplicate. Duplicate extracts were combined and concentrated by rotary-evaporation prior to radio-HPLC analysis.

Metabolite quantitation
The pooled urine, fecal extract and bile samples were analyzed by a radio-HPLC to quantitate radioactive metabolites.
Primary characterization/identification of radioactive metabolites was based on HPLC co-chromatography (co-injection) with the reference substances. To further identify/characterize radioactive metabolites, HPLC peaks of interest were isolated by a preparative HPLC. The isolated fractions were concentrated in vacuo, then analyzed by HPLC. The isolated radioactive metabolite was subjected to TLC co-chromatography (report section 4.12) with the reference substances and/or mass spectrometry to confirm its identity.

Radioassay
Liquid samples
Radioactivity in liquid sample was determined by LSC. Generally, duplicate or triplicate aliquots of a liquid sample were dissolved in either AtomlightTM, Pico-FluorTM plus or Hionic-FluorTM scintillation cocktail (all PerkinElmer Japan Co., Ltd.) prior to LSC Analysis.

Solid samples
Solid sample was subjected either solubilization (fecal homogenate) or oxidative combustion (homogenate of residual carcass, residues on a filter paper, etc.).

Detection limit for radio-analysis
For LSC analysis, the detection limit was fixed at 40 dpm/LSC sample which is approximately twice the background radioactivity. This means LSC output showing < 40 dpm was defined as “not detected”. The detection limit for HPLC analysis was defined as 4.66σ (σ= sqr(BKG×t) , BKG: rate of the background area counts (counts per minute), t: counting time (min)) based on HPLC background radio area counts. The background area counts were obtained from the HPLC region where radioactive peaks were not detected (e.g. HPLC area counts of 0 to 3 and 57 to 60 min for the HPLC method 2). HPLC detection limits were determined for individual runs.

Data expression and calculation
The quantity of radioactivity in a sample was expressed as percentage of the administered radioactivity (%AD) as shown below.

Percentage of the administered dose (%AD) = [(total radioactivity found in the sample, dpm) / (dosed radioactivity, dpm)] × 100

Details on excretion:

The material balance (sum of the excreted radioactivity in the urine, feces and remaining in the body and cage wash) ranged from 93.8% to 96.1% of the administered dose (%AD). Excretion into the urine and feces amounted to 5.48 to 7.23 %AD and 85.51 to 87.04 %AD, respectively for the low dose, and 19.44 to 20.40 %AD and 73.37 to 75.37 %AD, respectively for the high dose. The excreted radioactivity amounted to approximately 93.1 to 95.2%AD at 96 hr post dose. The results indicate fecal excretion is the predominant route for elimination of ME5343, irrespective of the dose level and sex. The radioactivity remaining in the gastro-intestinal tracts (with contents) and the carcass are 0.08 to 0.27 %AD and 0.68 to 0.93 %AD, respectively, at termination.

Biliary excretion
The material balance ranged from 94.3 % to 101.3 %AD. Excretion into the bile, urine and feces amounted to 53.10 to 53.28%AD, 12.96 to 16.23 %AD and 22.37 to 26.73 %AD, respectively for the low dose, and 40.16 to 40.45 %AD, 29.91 to 30.30 %AD and 24.05 to 26.36 %AD, respectively for the high dose. The excreted radioactivity amounted to approximately 92.3 to 97.1 %AD at 48 hr post dose. The estimated absorption rate (at 48 hr post dose), which was determined as the sum of the radioactivity found in the bile, urine and residual carcass, was 67 to 72 %. The results reveal that fecal excretion via biliary excretion is the predominant elimination route of orally administered test substance.

Metabolites identified:

yes

Remarks:

major metabolites (>5 %AD, either urine or feces) = ME5343-T1, ME5343-T2, ME5343-T8 and ME5343-T10

Details on metabolites:

Metabolites in bile
Unchanged [NCA-14C] lablelled test substance was not detected at significant level in the bile of the low and high dose experiments. Dose related difference was noted for the levels of ME5343-T17, which is more pronounced in the low dose (20.30 to 31.78 %AD) than that in the high dose (4.95 to 6.35 % AD). No significant sex related difference was found for the metabolite profile in the bile.

Metabolic pathway
Unchanged test substance was detected only in feces at a level of 10.41 to 38.69 %AD. The metabolites detected greater than 5 %AD in either urine or feces were ME5343-T1, ME5343-T2, ME5343-T8 and ME5343-T10. Minor metabolites, such as ME5343-T3 ME5343-T9, ME5343-T11, ME5343-T12 and ME5343-T17 were also detected with the level lower than 5 %AD.
In the bile sample, ME5343-T17, which is N-oxide of ME5343, was detected as the major metabolite followed by N-oxide of ME5343-T2, N-oxide of ME5343-T3, and ME5343-T1. Based on the metabolites identified/characterized as above, main metabolic reactions of ME5343 are considered to be 1) hydrolytic elimination of both 3- and 4-positions of cyclopropanecarbonyl groups to form the predominant metabolite, 2) hydroxylation at 4-methyl group, and 3) N-oxidation of pyridine ring. Hydrolytic elimination of the 3-cyclopropanecarbonyl group forming ME5343-T2 seems to be kinetically preferred over hydrolysis of the 4-cyclopropanecarbonyl group; hydrolysis of both esters produces ME5343-T1, the predominant metabolite. Hydroxylation of ME5343-T2 leads to ME5343-T8, and further hydrolysis produces ME5343-T9. In addition, particularly in bile, the N-oxidation of pyridine ring occurs to form ME5343-T17, and the hydrolytic products, N-oxide of ME5343-T2 and N-oxide of ME5343-T3. Dose related difference was noted for the levels of ME5343-T17, which is higher in the low dose (20.30 to 31.78 %AD) than that in the high dose (4.95 to 6.35 % AD). Levels of N-oxide metabolites were mainly not detected in the feces, so further metabolism of these components is occurring prior to or in conjunction with biliary excretion via the feces. No significant sex related difference was observed in excretion route and metabolic pathway of orally administered [NCA-14C] labelled test substance.

See also attached document - proposed metabolic pathway.

Concentration, homogeneity and stability of dosing suspensions

The radiochemical concentrations of [NCA-¹⁴C] labelled test substance in the low and high dose formulations were 0.800 to 0.896 MBq [test substance]/g (equivalent to 0.322 to 0.359 mg [test substance]/g) and 0.767 to 0.776 MBq [test substance]/g (equivalent to 30.9 to 31.3 mg [test substance]/g), respectively. The coefficient of variance (C.V.) in triplicate determinations of above ¹⁴C-concentrations was low as < 0.5 % indicating that the formulations were prepared as homogeneous. The radiochemical purities were found to be greater than 95% at the time of dose administration. All dose formulations were acceptable to use for the study.

The average levels of [NCA-¹⁴C] labelled test substance administered to individual rats were 3.0 to 3.1 mg/kg bw (7.48 to 7.60 MBq/kg bw) and 302 to 307 mg/kg bw (7.51 to 7.63 MBq/kg bw) for the low and high dose experiments, respectively, indicating that the administered weight and radioactivity of the test substance were adequate. The administered volume of the dose was 9.3 to 9.8 mL/kg bw.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From: 2010-06-30 To: 2015-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

other: pharmacokinetic

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF (12-Nousan-No.8147, 2-3-1)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

NON RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Meiji Seika Pharrna Co., Ltd., TA10224601
- Purity: 98.50 %

RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Institute of lsotopes Co., Ltd. (Hungary), XVII/53

RADIOLABELLING INFORMATION
- Radiochemical purity: 98.8 % by radio-HPLC
- Specific activity: 3.29 MBq/mg (1955 MBq/mmol)
- Locations of the label: nicotinic acid 9-14C

STORAGE CONDITIONS OF TEST MATERIAL
- The radiolabeled test substances were stored in a freezer set at -20 °C, and the unlabeled test substance was stored in a
refrigerator set at 2-10 °C.

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

Fischer rats are commonly used in animal metabolism studies. Fischer rats were used in toxicology studies with the test compound.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Stone Ridge, NY or Portage, MI)
- Age at study initiation: 9-10 weeks
- Housing: individually housed
- Diet: Harlan Teklad Certified Global Diet 2016 meal/pellets, fed ad libitum
- Water: fresh water, ad libitum
- Acclimation period: 1day (surgically prepared rats), 5 days (all other rats)

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: >10 times/hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2010-10-20 To: 2011-06-24

Route of administration:

oral: gavage

Vehicle:

other: Hydroxypropylcellulose (3%)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A stock solution of the radiolabeled test material was prepared by dissolving the entire portion of radiolabeled material in certified ACS grade ethyl acetate (50 mL). An aliquot was removed and measured by weight. The ethyl acetate was removed under nitrogen and the residue dissolved in Optima grade acetonitrile. Duplicate aliquots (10 µL) were removed from the dilution, mixed with Ultima Gold cocktail (Packard Instrument Co., Meriden, CT) and then analyzed for radiolabel content by LSC. The stock solution was stored in a freezer when not in use.

Dose solutions were prepared in 3% hydroxyl propyl cellulose (HPC) as follows. For both dose levels, an appropriate amount of unlabeled test substance and [NCA-14C] labeled test substance was added to each container. The organic solvent was removed by placing the containers under a stream of nitrogen gas. The formulation was brought up to the appropriate volume with vehicle (HPC). A magnetic stirring bar was added to the container and the dose was stirred vigorously. Three aliquots of each dosing suspension were taken for the determination of radiolabel concentration and homogeneity before and after dosing. Radiochemical purity was also determined before and after dosing.

The doses were homogeneous based on the LSC data. Radiochemical purity of each dose preparation was determined by HPLC. The dose formulations were prepared the day prior to dosing and stirred overnight at ambient temperature in the dark.

Duration and frequency of treatment / exposure:

single gavage

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

Pilot Pharmacokinetic study: 4 per sex per dose
Pharmacokinetic study: 5 per sex per dose
Tissue distribution study: 3 per sex per dose

Control animals:

no

Positive control reference chemical:

no

Details on study design:

Rats were assigned to the treatment groups using a computer-generated random permutation program. The rats were randomized to control bias in the study. The required number of experimental rats was consecutively chosen from the randomized list. The remaining rats were designated as replacement animals and were available for use in the event that a previously chosen animal was determined to be unsuitable for the treatment.

Animals were weighed immediately prior to dosing to determine the dose to be administered. The dose was delivered to the stomach of each rat via a gavage tube and syringe. Each syringe was weighed before and after dose administration to determine the actual dose administered. The administered dose was calculated as follows:
Weight of dose = (syringe weight before dosing – syringe weight after dosing)

The amount of test material and radiolabel administered to each animal was calculated as follows:
mg of the test substance administered to rat = (dpm administered to rat/specific activity/1000)
dpm administered to rat = (weight of dose, g)( dpm per gram of dose preparation)

Immediately after dose administration, each animal was placed into a separate metabolism cage.

See also Table 1.

Details on dosing and sampling:

SAMPLE COLLECTION AND ANALYSIS PROCEDURES
All samples collected during this study were placed in labeled containers. Each label contained the following information: 1) Study number, 2) Animal number, 3) Sample type and collection interval, and 4) Date of collection. Samples not analyzed immediately following collection, or remaining samples following analysis, were stored. Whole blood samples were stored refrigerated (~ 4 °C); while tissues were stored frozen (approximately -20 °C). Cage washes and solubilized samples were stored refrigerated (~4 °C) or frozen. Excreta samples (urine, feces) were stored frozen (approximately -20 °C).

Urine, Feces, and Cage Wash
Urine was collected from animals following the 14C dose administration. Urine was collected at 24-hour intervals until termination. For exposure periods of 24 hours or less than 24 hours in the tissue distribution experiment, urine was collected at termination. Urine from these experiments was not analyzed for radiolabel content. All urine samples were collected and pooled for disposal. Feces were collected from each animal from all studies.

Feces were collected at 24-hour intervals until termination. For exposure periods of 24 hours or less than 24 hours in the tissue distribution experiment, feces were collected at termination. Feces from these experiments were not analyzed for radiolabel content. All feces amples were collected and pooled for disposal.

After each urine and fecal collection, a cage wash was performed. When animals are present, cage washes consisted of water only. At termination, all cages were rinsed with water followed with a methanol rinse. The water and methanol rinses were combined from this final collection. Cage washes from these experiments were not analyzed for radiolabel content. All cage wash samples were collected and pooled for disposal.

Tissues, Blood, and Carcass
Animals used for the pharmacokinetic experiments had jugular cannulae surgically implanted prior to dose administration. For each animal in the pilot pharmacokinetic experiment, approximately 200 µL of blood was removed via the jugular cannula or orbital sinus at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 hours after dosing and placed in tubes containing K3EDTA anticoagulant. Two animals per sex per dose group were sampled alternately for each timepoint (6 samples from each animal) for the pilot pharmacokinetic experiment, due to blood volume constraints. For the pharmacokinetic experiment, blood samples (approximately 250 µL) were collected from each animal at 10 time points (0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours) and placed in tubes containing K3EDTA. Time points and intervals of blood samples collection for the pharmacokinetic study were determined by the pilot PK study. Following each sample collection the rats were infused with an equal volume of saline to maintain blood volume. For the pilot and plasma PK experiments, 50 µL of whole blood from each sample was placed in hematocrit tubes and centrifuged to separate plasma from packed cells. The packed cell volume was determined. The remaining blood sample was separated into packed cells and plasma. The plasma was then removed, weighed and analyzed directly by LSC of two aliquots. For the blood PK experiment two aliquots were analyzed by combustion followed by LSC to determine radioactivity levels in whole blood. In the pharmacokinetic experiments, no tissues were collected. Each of the following tissues were obtained from the animals from the tissue distribution experiment: whole blood, blood cells, plasma, spleen, thymus, mesenteric lymph nodes, bone marrow (femur), adrenal glands, pituitary gland, thyroid/parathyroid, bone (femur), skeletal muscle, heart muscle, brain, spinal cord, prostate gland (males), epididymis (males), testes (males), uterus (females), ovaries (females), eye, adipose tissue, skin with hair, kidneys, liver, urinary bladder, GI tract (with contents), lungs, and pancreas. Tissue samples (except blood) were stored at approximately -20 °C until analysis. Blood samples were stored at approximately 4 °C until analysis. The weight of each sample was determined. The radiolabel content in the tissue samples was determined by solubilization followed by LSC. These tissue samples were solubilized in entirety with 10% sodium hydroxide:methanol:Triton X, 8:1:1 (v/v/v) at approximately 60 °C. Duplicate aliquots of each solubilized tissue sample were analyzed by LSC, except for solubilization analysis of small tissues (adrenals, urinary bladder, bone marrow, eye, mesenteric lymph nodes, spinal cord, thymus, thyroid/parathyroid, epididymis, ovaries, prostate, and uterus) where the entire sample was analyzed in a single vial. A terminal blood sample was collected from each animal under CO2/O2 anesthetic via abdominal-aorta at each termination time. As much blood as possible was collected from each animal into a tube with anticoagulant. Whole blood samples were split into two portions. One portion was centrifuged to obtain plasma and packed cells. Aliquots of plasma from individual animals were removed and analyzed directly by LSC. Aliquots of packed cell from the individual animals were analyzed by combustion followed by LSC. The second portion of whole blood was stored in a refrigerator. Aliquots of whole blood from the individual animals were analyzed by combustion followed by LSC. The total weight of each carcass was recorded. The carcasses were stored at approximately -20 °C until they were analyzed. Carcasses from the tissue distribution experiments were analyzed in entirety. Aliquots of the solubilized carcasses were analyzed for radiolabel content by LSC.

Preliminary studies:

Pharmacokinetic parameters were calculated from the whole blood, plasma, and red blood cell total radioactivity concentration data. For the 3 mg/kg dose, the Tmax for whole blood and plasma was 1 hour for males and 0.5 hours for females, and the Tmax for red blood cells was 0.25 hours for males and 0.5 hours for females. For the 300 mg/kg dose, the Tmax for whole blood, plasma, and red blood cells was 4 hours for males and females.

Elimination half-lives were calculated by WinNonlin, allowing WinNonlin to maximize the R-squared value for the elimination phase. For the 3-mg/kg dose level, the elimination half-life was 5.0 hours for males and 2.1 hours for females for whole blood, 4.1 hours for males and 3.2 hours for females for plasma, and 3.6 hours for males and 2.4 hours for females for red blood cells. Elimination half-lives were longer for whole blood, plasma, and red blood cells for the 300-mg/kg dose than for the 3 mg/kg dose. For the 300-mg/kg dose level, the elimination half-life was 7.3 hours for males and 7.0 hours for females for whole blood, 6.8 hours for males and 6.0 hours for females for plasma, and 11.9 hours for males and 9.6 hours for females for red blood cells.

For whole blood and plasma, increases in Cmax with increasing dose were slightly less than dose proportional. For red blood cells, increases in Cmax with increasing dose were approximately dose proportional. For whole blood, plasma, and red blood cells, increases in AUC0-∞ with increasing dose were greater than dose proportional.

Within each matrix and dose level (whole blood, plasma, red blood cells), Cmax was generally similar between males and females. For the 3 mg/kg dose, AUC0-∞ was approximately 1.5-2-fold higher in males than females. For the 300 mg/kg dose, AUC0-∞ was similar in males and females

Details on absorption:

Pharmacokinetic parameters were calculated from the mean whole blood, plasma, and red blood cell total radioactivity concentration data. For the 3 mg/kg dose, the Tmax values for whole blood, plasma, and red blood cells were 1.0, 0.5, and 1.0 hours, respectively, for males, and 0.5, 0.5, and 0.25 hours, respectively, for females. Tmax was later for the 300 mg/kg dose than for the 3 mg/kg dose. For the 300-mg/kg dose, the Tmax values for whole blood, plasma, and red blood cells were 4.0, 4.0, and 4.0 hours, respectively, for males, and 2.0, 2.0, and 2.0 hours, respectively, for females.

Elimination half-lives were calculated by WinNonlin, allowing WinNonlin to maximize the R-squared value for the elimination phase. For the 3 mg/kg dose level, the elimination half-life values were 2.5 hours for males and 1.0 hour for females for whole blood, 4.7 hours for males and 4.8 hours for females for plasma, and 2.1 hours for males and 1.2 hours for females for red blood cells. Elimination half-lives were longer for whole blood, plasma, and red blood cells for the 300 mg/kg dose than for the 3 mg/kg dose. For the 300-mg/kg dose level, the elimination half-life values were 16.0 hours for males and 15.1 hours for females for whole blood, 10.2 hours for males and 7.9 hours for females for plasma, and 31.4 hours for males and 43.6 hours for females for red blood cells.

For whole blood, plasma, and red blood cells, increases in Cmax with increasing dose were greater than dose proportional; for a 100-fold increase in dose from 3 to 300 mg/kg, Cmax increased 147-fold for males and 287-fold for females for whole blood, 130-fold for males and 245-fold for females for plasma, and 165-fold for males and 363-fold for females for red blood cells. For whole blood, plasma, and red blood cells, increases in AUC0-∞ with increasing dose were also greater than dose proportional. For a 100-fold increase in dose from 3 to 300 mg/kg, AUC0-∞ increased 760-fold for males and 1603-fold for females for whole blood, 494-fold for males and 883-fold for females for plasma, and 1661-fold for males and 3730-fold for females for red blood cells. Within each matrix and dose level (whole blood, plasma, red blood cells), Cmax was generally similar between males and females. For the 3 mg/kg dose, AUC0-∞ was approximately 2.2- to 2.6-fold higher in males than females. For the 300 mg/kg dose, AUC0-∞ was similar in males and females.

Details on distribution in tissues:

For the 3 mg/kg dose level, the Tmax for most tissues was 0.5 hours, the first sampling point postdose, except for bone marrow and GI tract and contents, where the Tmax was 8 hours. Tissues with mean concentrations at Tmax that exceeded 0.250 μg-equiv/g were GI tract and contents, liver, adrenals, kidney, lung, pancreas, prostate, bone marrow, mesenteric lymph nodes, parathyroid/thyroid, and heart. By 96 hours max postdose, tissue concentrations were less than 0.03 μg-equiv/g in all tissues.

For the 300 mg/kg dose level, the Tmax for all tissues was 2 hours, the first sampling point postdose. Tissues with mean concentrations at Tmax that exceeded 40 μg-equiv/g were 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. By 96 hours postdose, tissue max concentrations were less than 7 μg-equiv/g in all tissues.

For the 3 mg/kg dose level, the mean percent recovery of administered dose in tissues and carcass was 63.85 % for males and 69.81 % for females at the 0.5-hour termination time, 66.28 % for males and 68.20 % for females at the 8-hour termination time, and 0.29 % for males and 0.24 % for females at the 96-hour termination time. Tissues with the highest percent of administered dose at the 0.5- and 8-hour termination times were GI tract and contents, liver, and residual carcass.

For the 300 mg/kg dose level, the mean percent recovery of administered dose in tissues and carcass was 95.78 % for males and 104.08 % for females at the 2-hour termination time, 64.04 % for males and 49.13 % for females at the 24-hour termination time, and 0.80 % for males and 0.79 % for females at the 96-hour termination time. 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.

Concentration, homogeneity and stability of dosing suspensions

Radiochemical purity values (before dosing) were >95% for the 3-mg/kg dose level and the 300-mg/kg dose level. The results of the radiochemical purity analyses after dosing indicate that radio purity of the dose preparations was 95% or above during the time of dose administration.

Table 2 Dose administered

Experiment	Dose level (mg/kg)	Chemical concentration (mg/g)	Average radiochemical purity (%)	Specific activity (dpm/μg)
P-PK	3	0.30	97.12	146,914
	300	30.00	96.87	1,414
PK	3	0.31	97.51	148,455
	300	30.00	96.77	1,432
TD	3	0.36	95.48	156,032
	300	29.97	95.32	1,282

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From: 2011-08-05 To: 2015-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan/MAFF: Guidelines on the Compiling of Test Results on Toxicity; Tests on In Vivo Fate In Animals

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

NON RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: COD-001545 (purity 97.2 %) and 36/55-097-4 (purity 99.9 %)

RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material, locations of the label:
1021-0101 (pyranone-4-C14, radiochemical purity: >95 %, Specific activity 3.43 (MBq/mg))
1021-0204 (pyranone-4-C14, radiochemical purity: >98.0 %, Specific activity 3.67 (MBq/mg))
1022-0101 (pyranone-4-C13,chemical purity: 100.0 %, Specific activity 3.67 (MBq/mg))

STABILITY TEST MATERIAL
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 97633 Sulzfeld, Germany
- Age at study initiation: at least 8 weeks
- Housing: up to 5 animals during the acclimatization period; single housing in metabolism cages during the experiment
- Diet: Provimi Kliba SA, 4303 Kaiseraugst, Switzerland ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days before the beginning of the experimental phase

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 15 changes/hr
- Photoperiod: 12-hour light and dark cycles

IN-LIFE DATES: From: 2011-08-09 To: 2014-07-20

Route of administration:

other: oral: gavage and intravenous

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
In order to achieve the required specific activity appropriate amounts of the radio-labeled testsubstance solution were taken and the organic solvent (acetonitrile (ACN)) was evaporated to dryness. Unlabeled material was evaporated to dryness when added as a solution (for low dose groups stock solutions of unlabelled test substance were prepared separately). The aqueous vehicle (0.5% carboxymethylcellulose (CMC) in tap water) was added to the test substance(s). The high dose group of tissue distribution resulted in inconsistent data. These results were attributed to changes of viscosity of the test-substance preparation over time in combination with the technical set up in this experiment when 0.5 % CMC in tap water was used as a carrier. Hence, 1 weight% Cremophor was added to the carrier (0.5 % CMC in tap water) to guarantee the consistency of the test-substance preparation when the experiment was repeated. Due to the possibility to facilitate structure eludication of formed metabolites in bile excretion experiments, 13C-labeled test substance was mixed with non-labeled test substance in a ratio of 1:2.5 (w:w) for the test-substance preparation of the male animals of the high dose group. For this test-substance preparation, appropriate amounts of a solution of the 13C-labelled tests ubstance were taken and the organic solvent (ACN) was evaporated to dryness. Respective amounts of the unlabeled material and the aqueous vehicle (0.5 % carboxymethylcellulose (CMC) in tap water] were added. The preparations were homogenized by ultra-sonification and continious stirring. At least before start and at the end of the administration, samples were taken to determine the amount of radioactivity in the preparations to demonstrate the correct concentration of the test substance, its homogeneity and the radiochemical purity of prepared 14C labelled test substance.

Duration and frequency of treatment / exposure:

Blood/Plasma Concentration: 1 oral dose or 1 intavenous dose
Balance/Excretion: 1 oral dose or 15 days repeated oral dose
Tissue distribution: 1 oral dose
Bile Excretion: 1 oral dose

See also Table 1.

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

oral: gavage

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

oral: gavage

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

oral: gavage

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

intravenous

No. of animals per sex per dose / concentration:

Blood/Plasma Concentration: 4 per sex per dose
Balance/Excretion: 4-5 per sex per dose
Tissue distribution: 12 per sex per dose
Bile Excretion: 6 males and 12 females per dose

Control animals:

no

Positive control reference chemical:

no

Details on study design:

Administration of test material
10 mL/kg body weight of a preparation was administered orally to rats by gavage. 1 mL/kg body weight of a preparation was administered to rats intravenously by syringe into the tail vein.

Clinical exams and Body Weight
A check for moribund and dead animals was conducted at least once daily. A cage side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

The body weight was determined on the days of administration prior to dosing in the plasmakinetic and the balance experiments. In the bile experiments the body weight was determined on the first day of Baytril® administration.

Details on dosing and sampling:

Blood/Plasma concentration (Experiments 1,2,10, 11)
Animals were placed in steel wire mesh cages after treatment with 14C-BAS 440 I and blood samples (100 – 200 μL) were taken from the retroorbital sinus under isoflurane anaesthesia at the following time points or by exanguination (under isoflurane anaesthesia) at the last time point: 1; 2; 4; 8; 24; 48; 72; 96,120, 144 and 168 hours. For experiment 11 (intravenous dose group) blood samples were taken additionally directly after administration and after 0.5 hours.

Balance / Excretion (Experiments 3, 4, 5)
In this set of experiments, animals were dosed and then placed in metabolism cages in order to collect urine after 6, 12 and 24 hours and subsequently in time intervals of 24 hours up to 168 hours and feces in intervals of 24 hours up to 168 hours. In the balance experiment 4, the first two male animals were placed in closed metabolism cages in order to additionally collect exhaled air for 48 h. Since < 2 % of the total radioactive dose were detected in exhaled air, all experiments were carried out in open systems. After 168 hours, animals were sacrificed and the following organs/tissues were checked for remaining radioactivity:
heart, liver, spleen, bone, skin, lung, ovaries, carcass, muscle, kidney, testes, brain, pancreas, uterus, adipose tissue, stomach & stomach contents, thyroid gland, adrenal glands, blood cells and plasma, gut and gut contents, bone marrow.

For balance estimates the cage wash was also checked for radioactivity.

Tissue distribution (Experiments 6 and 7)
In this set of experiments animals were treated and then placed in steel wire mesh cages. Three animals were sacrificed at 4 defined time points after dosing that were selected based on the results of the blood and plasma kinetic experiments. The time points selected correspond to the following time points in plasmakinetics: maximum plasma concentration MPC, 1/2 MPC, 1/4 MPC and 1/8 MPC at the high and low dose level.

During necropsy, gel-like agglomerations of the test-substance preparation were observed in the stomach of selected animals of the high dose group which were assessed to be responsible for inconsistent results of plasma levels of this dose group. These results were attributed to changes of viscosity of the test-substance preparation over time in combination with the technical set up in this experiment when 0.5 % CMC in tap water was used as a carrier (although the test-substance preparation associated analytics yielded valid results and dosing of the animals by gavage was without unexpected findings). Hence, 1 weight% Cremophor was added to the carrier (0.5 % CMC in tap water) to guarantee the consistency of the test substance preparation when the experiment was repeated. The organs/tissues of the first experiment of the high dose group were not worked up.
After sacrifice remaining radioactivity was measured in the following organs and tissues:
heart, bone, blood cells and plasma, liver, muscle, pancreas, spleen, kidney, thyroid, brain, carcass, adrenal glands, skin, adipose tissue, gut and gut contents, lung, testes, stomach and stomach contents, uterus ovaries, bone marrow

Excretion via bile (Experiments 8 and 9)
In this experiment the bile duct of rats was cannulated in a surgery. The surgery was performed under isoflurane anesthesia and analgesia. For analgesia, Buprenorphin® was given before surgery (at least 30 minutes before surgery started) and on the following days until 1 day before test substance administration. The dose of Buprenorphin® was about 0.01 mg/kg bw and day. For antibiosis animals were treated with 1 % Baytril® approx. 24 hours before surgery and on the following days after the surgery until 1 day before administration. In addition about 1 mL/100g bw of 0.05 % Baytril® was administered to the animals during surgery. A second catheter was put in the duodenum to supplement the animal with a solution of synthetic bile acids by continuous infusion. The bile catheterized animals were placed in Polycarbonate type M III cages until administration. To guarantee a permanent correct technical set up, animals were checked regularly during the experiment and if necessary, the technical equipment was rearranged (e.g. distorted catheters). For this purpose, animals were kept under isoflurane anesthesia if necessary.

Before administration, the cannulated animals were placed in metabolism cages in order to collect bile in three hour intervals as well as urine and feces in 24 hour intervals up to 72 hours, depending on the health state of the animals and the excretion rate. After 72 hours, animals were sacrificed and defined samples were checked for remaining radioactivity:
bile, urine and feces of defined collection intervals, stomach and stomach contents, gut and gut contents, carcass.

For balance estimates the cage wash was also checked for radioactivity.

Description of analytical procedures
Homogeneity / concentration control and stability analysis
The amounts of radioactivity of 14C labeled test substance in the test-substance preparations were determined in samples that were taken at least before and after dosing. Analyses of these samples allowed to demonstrate the homogeneity, correctness of the concentration and the stability of the test substance in the test-substance preparations.

The analyses of the test-substance preparation were performed by HPLC

Total radioactivity in biological matrices
After weighing, aliquots of liquid samples (bile, urine, plasma and cage wash) were mixed with scintillation cocktail (Hionic Fluor, Perkin Elmer) and analyzed for radioactivity without any additional treatment. Soluene®-350 (Perkin Elmer) was added to blood cells. The samples were incubated followed by the addition of isopropanol. Then the samples were bleached by the addition of perhydrol solution (30 % H2O2). After further incubation, Hionic Fluor cocktail (Perkin Elmer) was added and the samples were measured by liquid scintillation counting. Feces, contents of gut and stomach, carcass, lung, heart, spleen, kidney, testes, brain and liver were suspended in deionized water and were homogenized using a WARING Blender. Aliquots of the suspensions of feces, contents of gut and stomach and carcass were dried by lyophylisation, dissolved with Soluene®-350, filled up with isopropanol, bleached with perhydrol solution (30 % H2O2) and Hionic Fluor was added after incubation before measurement of radioactivity by LSC. Feces samples of early collected fractions of balance experiments up to 72h and of the low dose group were worked up by diluting the samples in an appropriate solvent followed by addition of Hionic Fluor and LSC measurement. Prepared aliquots of suspensions of lung, heart, spleen, kidney, testes, brain and liver and whole samples of muscle, adipose tissue, uterus/ovaries, thyroid gland, adrenal gland, pancreas, skin, bone marrow, stomach and gut Soluene®-350 and isopropanol were added. Then the resulting solutions were bleached with perhydrol solution (30 % H2O2) and Hionic Fluor was added before measurement of radioactive residues by LSC. Bone samples were treated with hydrochloric acid (4M) and Hionic Fluor was added before measurement of radioactivity by LSC.

Details on absorption:

Mean plasma concentrations of radioactivity after single oral administration of 14C labelled test substance to male and female rats at target dose levels of 300, 30 and 3 mg/kg bw and after single intravenous administration of 14C labeled test substance to male and female rats at a target dose level of 0.5 mg/kg bw are presented in Table 2. The calculated kinetic data are summarized in Table 3.

Details on distribution in tissues:

Mean results of tissue distribution of radioactivity at defined time points after a single oral administration of 14C labelled test substance to male and female rats at target dose levels of 300 and 3.0 mg/kg bw are presented in Table 4 and Table 5.

Details on excretion:

Balance / excretion
Overall, the balance data demonstrate that major excretion of 14C labelled test substance dosed orally by gavage to Wistar rats occurred via the feces with mean values of 74.34 and 74.49 as well as 72.41 and 78.19 % of dose for male and female animals of the single high as well as the multiple (14+1) high dose groups, respectively. The mean total amount of radioactivity excreted via feces was found to be 85.87 and 87.43% of dose for male and female rats after single oral dosing of 3 mg/kg bw 14C labelled test substance (low dose), respectively. Excretion via feces accounted for 64.96 to 88.49 % of dose for single animals of the high dose of 300 mg/kg bw (single dose and multiple dose (14 +1)) and to 79.64 to 93.08 % of dose for the single animals of the low dose group.

Urinary excretion was a minor excretion pathway with mean values of 20.19 and 21.23 as well as 17.91 and 15.44 % of dose for male and female animals of the single high as well as the multiple (14+1) high dose groups, respectively. The mean total amount of radioactivity excreted via urine was found to be 5.49 and 5.91 % of dose for male and female rats after single oral dosing of 3 mg/kg bw 14C labelled test substance (low dose), respectively. Urinary excretion of 14C labelled test substance after oral dosing accounted to 16.44 to 29.86 % of dose for single animals of the high dose of 300 mg/kg bw (single dose), to 13.51 to 23.45 % of dose for single animals of the high dose of 300 mg/kg bw (multiple dose) and to 4.04 to 8.45 % of dose for the single animals of the low dose group.

The pattern of excretion after repeated oral administration (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) showed slightly lower amounts of radioactivity excreted in urine than in the single dose experiment, giving an indication that changes in kinetics / metabolism may occur after multiple dosing. Excretion was rapid and occurred to a major extent within two days after dosing.

Table 6 shows the mean values of excreted and residual radioactivity after single oral administration of 14C labelled test substance to male and female rats at target dose levels of 300 and 3 mg/kg bw and after 15 daily oral administrations of the test substance(14 x unlabeled at 300 mg/kg bw; 1 x labeled at 300 mg/kg bw at day 15).

Bile Excretion
The mean biliary excretion of radioactivity after a single oral administration of 14C labelled test substance to rats at target dose levels of 300 and 3 mg/kg bw is presented in Table 7 and Table 8.

Oral absorption of 14C labelled test substance in rats was more or less dose independent and was calculated to about 57 % and 60 % of the administered dose for males and females at a dose level of 300 mg/kg bw and about 57 % of the applied dose for both sexes at a dose level of 3 mg/kg bw.

Metabolites identified:

no

Concentration, homogeneity and stability of dosing suspensions

The stability of the test substance in the test-substance preparations over the test period was verified by analyses. The concentrations of the test substance in the test-substance preparations were confirmed by analyses. The homogeneous distributions of the test substance in the test-substance preparations were demonstrated by a low standard deviation in the concentration control analyses of generally < 5 %. For experiment 7 (tissue distribution), concentration control analyses revealed a standard deviation of 9.8 % which is still assessed to yield acceptable homogeneity within the precision of the method. The measured specific activities were generally in accordance with their nominal values.

Clinical examinations

Overall, the experiments of the current study demonstrate that the test substance does not induce mortality to Wistar rats up to an oral dose of 300 mg/kg bw for up to 2 weeks. An intravenous dose of 0.5 mg/kg bw was well tolerated by the animals. There were three animals that died during the course of the study. One death (high dose) was due to a gavage error. The remaining animals that died were in the low dose groups, and the deaths were judged to be not related to treatment.

No abnormal body weight change was observed.

Upon necropsy gel-like agglomerates of the test-substance preparation were observed in the stomach of select animals of the high dose group of the tissue distribution experiment. This finding was correlated with inconsistent data for plasma concentrations at defined time points. Therefore this experiment was repeated

Table 2: Mean plasma concentration of radioactivity after single administration of ¹⁴C labelled test substance at dose levels of 300, 30 and 3 mg/kg bw (oral) and 0.5 mg/kg bw (iv) to male and female rats [μg Eq/g plasma] 

Time	300 mg/kg bw		30 mg/kg bw		3 mg/kg bw		0.5 mg/kg bw
[h]	Males	Females	Males	Females	Males	Females	Males	Females
0	---	---	---	---	---	---	1.52	0.65
0.5	---	---	---	---	---	---	0.30	0.23
1	28.34	29.25	6.24	5.97	0.39	0.40	0.21	0.15
2	34.70	40.90	5.15	7.14	0.21	0.19	0.14	0.11
4	45.66	61.11	3.17	8.75	0.07	0.06	0.11	0.09
8	30.62	46.20	1.65	11.83	0.05	0.06	0.07	0.07
24	9.28	8.87	0.11	9.92	0.01	0.01	0.03	0.03
48	0.61	1.54	0.06	3.55	0.01	0.01	0.02	0.02
72	0.35	0.42	0.05	1.68	0.01	0.00	0.02	0.01
96	0.31	0.29	0.03	0.92	0.00	0.00	0.01	0.01
120	0.30	0.28	0.03	0.54	0.00	0.00	0.01	0.01
144	0.30	0.26	0.03	0.34	0.00	0.00	0.01	0.00
168	0.19	0.16	0.02	0.23	0.00	0.00	0.00	0.00

Table 3: PK parameters of radioactivity in plasma after single oral administration of ¹⁴C labelled test substance at dose levels of 300, 30 and 3 mg/kg bw to male at female rats 

Sex	Dose [mg/kg bw ]	Cmax [μg Eq/g]	Tmax [hour]	Initial half life [hour]	terminal half life [hour]	AUC0-168 [μg Eq *hour/g]	AUC0-∞ [μg Eq *hour/g]
male	300, p.o.	45.66	4	7.14	92.02	756.3	783.6
	30, p.o.	6.24	1	3.63	82.88	47.2	49.7
	3, p.o.	0.39	1	1.22	18.7	2.1	2.1
	0.5, i.v.	1.52	directly1	0.34	61.91	4.1	4.5
(AUC/dose)3mg/kgbw p.o./ (AUC/dose)0.5mg/kgbw i.v. : 9.0 % (AUC/dose)300mg/kgbw p.o./ (AUC/dose) 0.5mg/kgbw i.v.: 33.8 %
female	300, p.o.	61.11	4	8.16	81.57	982.1	1003.3
	30, p.o.	11.83	8	18.74	38.98	531.1	543.9
	3, p.o.	0.4	1	1.11	16.8	2.2	2.2
	0.5, i.v.	0.3	directly1	0.46	48.68	3.6	3.8
AUC/dose)3mg/kgbw p.o./ (AUC/dose)0.5mg/kgbw i.v. : 11.2 % (AUC/dose)300mg/kgbw p.o./ (AUC/dose)0.5mg/kgbw i.v. : 50.8% 1means directly after administration

Table 4: Mean tissue concentration of radioactivity after single oral administration of ¹⁴C labelled test substance at a dose level of 300 mg/kg bw to male and female rats (ug Equiv /g tissue) 

	Male				Female
Time after administration (hours)	4	12	24	36	4	14	20	36
Blood cells	14.20	5.40	5.73	1.05	16.00	8.86	11.71	7.08
Plasma	17.91	6.56	6.49	0.26	17.85	7.11	7.93	3.17
Lung	28.61	12.29	11.79	1.12	26.80	13.52	14.86	6.78
Heart	30.85	14.20	17.49	1.43	33.03	14.30	15.27	9.36
Spleen	25.45	9.95	11.64	0.77	26.05	11.25	14.29	6.23
Kidney	46.01	21.54	23.81	0.74	38.31	18.38	19.29	8.31
Adrenal glands	99.49	40.43	42.91	0.98	89.06	42.01	43.74	20.16
Testes/Ovaries	10.82	4.17	4.10	0.39	22.79	10.51	10.22	4.94
Uterus	---	---	---	---	32.68	13.81	13.78	8.90
Muscle	23.70	9.56	9.51	0.38	23.13	8.84	9.37	4.13
Brain	2.28	1.77	1.26	0.51	2.17	1.15	1.63	0.71
Adipose tissue	39.28	18.63	21.25	0.46	35.64	12.85	14.99	7.49
Bone	10.90	5.57	3.27	0.34	6.93	3.86	3.49	1.21
Bone marrow	26.86	11.95	12.22	3.19	23.96	12.54	9.70	10.02
Thyroid	54.90	25.50	29.38	5.36	60.66	23.71	26.04	18.50
Pancreas	45.60	21.74	24.47	0.95	45.26	19.74	19.89	8.24
Stomach content	3760.06	3993.00	3307.81	0.55	4762.89	3000.79	2560.71	1873.81
Stomach	1170.47	577.91	572.83	2.63	768.31	592.95	296.54	407.53
Gut content	3157.81	1779.02	1128.98	136.38	3955.58	2539.97	2044.09	820.40
Gut	170.89	104.63	105.60	5.85	182.15	139.54	78.66	43.81
Liver	100.69	49.81	63.01	3.15	81.49	45.59	52.73	22.96
Skin	21.78	8.74	10.38	0.89	22.81	9.72	9.48	4.18
Carcass	31.54	12.80	13.10	2.47	21.63	26.00	14.04	7.77

Table 5: Mean tissue concentration of radioactivity after single oral administration of ¹⁴C labelled test substance at a dose level of 3 mg/kg bw to male and female rats (ug Equiv /g tissue) 

	male				f emale
Time af ter administration (hours)	1	2	3.5	8	1	2	3.5	8
Blood cells	0.12	0.11	0.09	0.01	0.19	0.15	0.05	0.02
Plasma	0.28	0.25	0.16	0.02	0.24	0.17	0.05	0.01
Lung	0.45	0.44	0.29	0.04	0.41	0.30	0.08	0.02
Heart	0.71	0.59	0.37	0.11	0.50	0.37	0.08	0.05
Spleen	0.55	0.48	0.32	0.03	0.46	0.32	0.12	0.02
Kidney	1.18	1.24	1.08	0.10	0.85	0.57	0.15	0.03
Adrenal glands	2.96	2.68	1.69	0.33	1.90	1.25	0.27	0.11
Testes/Ovaries	0.08	0.14	0.13	0.04	8.30	4.84	8.44	0.40
Uterus	---	---	---	---	0.81	0.65	0.74	0.06
Muscle	0.36	0.39	0.28	0.04	0.36	0.30	0.09	0.02
Brain	0.03	0.03	0.03	0.02	0.03	0.02	0.01	0.00
Adipose tissue	0.50	0.91	0.83	0.09	0.56	0.62	0.25	0.06
Bone	0.13	0.20	0.16	0.01	0.17	0.13	0.04	0.01
Bone marrow	0.40	0.35	0.25	0.05	0.40	0.30	0.10	0.05
Thyroid	1.14	1.46	0.82	0.38	0.83	0.66	0.52	0.12
Pancreas	1.04	1.05	0.65	0.10	1.79	0.80	0.23	0.06
Stomach content	78.12	71.36	5.53	9.05	107.01	93.35	41.64	4.99
Stomach	15.76	20.85	2.60	1.09	18.90	35.67	6.18	0.82
Gut content	14.29	30.01	43.89	39.45	16.88	26.42	41.17	44.21
Gut	4.53	6.17	12.03	2.05	14.42	15.01	14.82	3.77
Liver	4.61	3.63	2.49	0.44	4.50	3.04	0.78	0.31
Skin	0.36	0.37	0.25	0.04	0.31	0.31	0.09	0.02
Carcass	0.36	0.44	0.34	0.06	0.45	0.40	0.21	0.04

Table 6: Mean excretion and retention of radioactivity after single oral administration of ¹⁴C labelled test substance at dose levels of 300 and 3 mg/kg bw and after 15 daily oral administrations of the test substance (14X unlabeled 300 mg/kg and 1X labeled 300 mg/kg bw at day 15) to male and female rats. Results are in % of dose administered 

Balance/Excretion	300 mg/kg bw		3.0 mg/kg bw		(14+1) 300 mg/kg bw
	male	female	male	female	male	female
Urine 0-6h	2.57	4.27	3.88	4.70	4.74	3.74
Urine 6-12h	5.34	6.03	0.80	0.49	6.82	4.97
Urine 12-24h	7.20	5.54	0.49	0.42	4.93	4.84
Urine 24-48h	4.53	4.02	0.23	0.17	0.74	1.59
Urine 48-72h	0.23	1.06	0.04	0.07	0.21	0.14
Urine 72-96h	0.11	0.13	0.02	0.03	0.12	0.07
Urine 96-120h	0.08	0.08	0.02	0.02	0.14	0.04
Urine 120-144h	0.07	0.05	0.01	0.01	0.12	0.03
Urine 144-168h	0.05	0.05	0.01	0.01	0.09	0.03
Subtotal Urine	20.19	21.23	5.49	5.91	17.91	15.44
Feces 0-24h	22.80	16.89	58.54	77.45	32.96	18.66
Feces 24-48h	42.15	32.50	24.73	7.91	36.32	51.68
Feces 48-72h	6.40	21.19	2.35	0.58	2.72	7.03
Feces 72-96h	0.43	3.18	0.16	1.43	0.16	0.66
Feces 96-120h	1.12	0.53	0.05	0.02	0.19	0.09
Feces 129-144h	0.05	0.15	0.02	0.01	0.04	0.04
Feces 144-168h	1.38	0.05	0.04	0.02	0.03	0.03
Subtotal Feces	74.34	74.49	85.87	87.43	72.41	78.19
Cage w ash	0.15	0.25	0.06	0.32	0.10	0.12
Blood cells	0.03	0.02	0.01	0.01	0.02	0.01
Plasma	0.00	0.00	0.00	0.00	0.00	0.00
Lung	0.00	0.00	0.00	0.00	0.00	0.00
Heart	0.00	0.00	0.00	0.00	0.00	0.00
Spleen	0.00	0.00	0.00	0.00	0.00	0.00
Kidney	0.00	0.00	0.00	0.00	0.00	0.00
Adrenals	0.00	0.00	0.00	0.00	0.00	0.00
Testes/Ovaries	0.00	0.00	0.00	0.00	0.00	0.00
Uterus	0.00	0.00	0.00	0.00	0.00	0.00
Muscle	0.00	0.00	0.00	0.00	0.00	0.00
Brain	0.00	0.00	0.00	0.00	0.00	0.00
Adipose Tissue	0.00	0.00	0.00	0.00	0.00	0.00
Bone	0.00	0.00	0.00	0.00	0.00	0.00
Bone marrow	0.00	0.00	0.00	0.00	0.00	0.00
Thyroid	0.00	0.00	0.00	0.00	0.00	0.00
Pancreas	0.00	0.00	0.00	0.00	0.01	0.01
Stomach cont.	0.00	0.00	0.00	0.00	0.00	0.00
Stomach	0.00	0.00	0.00	0.00	0.00	0.00
Gut cont.	0.01	0.01	0.01	0.01	0.01	0.01
Gut	0.01	0.01	0.03	0.01	0.01	0.00
Liver	0.02	0.01	0.01	0.00	0.02	0.02
Skin	0.07	0.04	0.02	0.02	0.10	0.01
Carcass	0.13	0.13	0.09	0.12	0.14	0.16
Total	94.96	96.20	91.60	93.84	90.74	94.00

Table 7: Excretion and retention of radioactivity via urine, feces and bile after single oral administration of ¹⁴C labelled test substance to male and female rats at dose levels of 300 and 3 mg/kg bw. (Results expressed as % of the radioactivity administered) 

	300 mg/kg bw		3.0 mg/kg bw
	Male	Female	Male	Female
Animal weight (g)	294.8	239.6	282.3	214.5
Spec Activity [MBq/g]	18.26	20.04	1879	1949
Dose admin. [mg/kg/bw]	286.6	322.3	3.13	3.10
Radioact. Dose [MBq/animal]	1.55	1.56	1.66	1.31
Urine 0-24h	13.31	17.31	17.20	10.26
Urine 24-48h	1.74	3.58	0.16	0.61
Urine 48-72h	0.13	0.59	0.06	0.06
Subtotal Urine	15.18	21.49	17.43	10.92
Feces 0-24h	15.37	4.17	24.67	16.81
Feces 24-48h	11.42	14.67	4.85	8.80
Feces 48-72h	5.18	3.80	0.49	6.08
Subtotal Feces	31.97	22.65	30.01	31.68
Cage wash	0.45	1.74	0.21	0.22
Stomach cont.	0.01	0.04	0.00	0.00
Stomach	0.00	0.01	0.00	0.00
Gut Cont.	0.14	0.38	0.03	0.19
Gut	0.04	0.03	0.32	0.02
Carcass	0.23	0.21	0.14	0.14
Subtotal Bile	41.30	36.16	39.16	45.49
Total	89.33	82.71	87.31	88.68
Bioavailability	57.16	59.61	56.94	56.77

Table 8: Excretion pattern of radioactivity via bile after single oral administration of ¹⁴C labelled test substance to male and female rats at dose levels of 300 and 3 mg/kg bw. (Results expressed as % of the radioactivity administered) 

	300 mg/kg bw		3 mg/kg bw
Time interval [h]	Males	Females	Males	Females
0-3	1.94	1.06	23.03	25.16
3-6	4.09	2.35	10.69	13.70
6-9	5.79	3.02	2.45	2.70
9-12	4.82	3.49	1.03	1.04
12-15	3.53	3.28	0.66	0.54
15-18	3.09	3.30	0.40	0.34
18-21	2.83	2.59	0.26	0.29
21-24	3.42	2.13	0.17	0.37
24-27	3.53	2.83	0.11	0.46
27-30	3.57	2.55	0.10	0.29
30-33	1.71	2.15	0.07	0.23
33-36	0.99	1.64	0.05	0.15
36-39	1.26	1.52	0.03	0.10
39-42	0.68	1.48	0.03	0.08
42-45	0.24	0.93	0.01	0.08
45-48	0.11	0.95	0.01	0.04
48-51	0.05	0.74	0.01	0.04
51-54	0.06	0.32	0.01	0.03
54-57	0.04	0.14	0.01	0.02
57-60	0.03	0.10	0.01	0.02
60-63	0.02	0.07	0.01	0.02
63-66	0.02	0.04	0.01	0.02
66-69	0.03	0.04	0.00	0.01
69-72	0.04	0.03	0.00	0.01
Total	41.89	36.75	39.17	45.72

Reference 6

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From: 2011-11-17 To: 2015-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Residue Chemistry Test Guidelines OPPTS 860.1000: Background (USA)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF Testing Guidelines for Toxicology Studies: Metabolism Animals (Japan)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Commision Directive 87/302/EEC of 18 November 1987, Part B, Toxicokinetics (EU)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

NON RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 36/55-097-4 (purity 99.9 %) and L82-65 (purity 99.8 %)

RADIOLABELED TEST SUBSTANCE

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material, location of the label:
1021-0101 (pyranone-4-C14, radiochemical purity: 98.6 %, chemical purity: 98.5%, specific activity 3.43 (MBq/mg))
1021-0101 (pyranone-4-C14, radiochemical purity: 98.7 %, chemical purity: 90.7%, specific activity 3.47 (MBq/mg))
1022-0101 (pyranone-4-C13,chemical purity: 100.0 %)
1022-2002 (pyranone-4-C13,chemical purity: 98.3 %)

STABILITY TEST MATERIAL
- The analytical investigations performed demonstrated the specific radioactivity, purity, stability and homogeneity of radiolabeled-BAS 440 I in the treatment preparations used for dosing within that study (dose groups V and W).

Radiolabelling:

yes

Species:

rat

Strain:

other: Wistar / Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: approximately 10-16 weeks at start of acclimatization period
- Weight at study initiation: 284 – 313 g (m), 242 – 266 g (f)
- Housing: Acclimatization: Group housing in macrolon cages (type 5). During the experiments: Single housing in macrolon cages (type 3).
- Diet: Pelleted standard Kliba 3433, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 – 22 °C
- Humidity: 45 – 55 %
- Air changes: > 30 changes/hr
- Photoperiod: Alternating 12-hour light and dark cycles

IN-LIFE DATES: From: 2012-10-22 To: 2012-10-30

Route of administration:

other: oral (gavage) and intravenous

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For oral administration of the test item to the animals of dose groups V and W, mixtures of 14C-labelled, 13C-labelled and unlabeled test substance were prepared in a ratio of approximately 48 : 15 : 37 (dose group V) or 8 : 26 : 66 (dose group W). 0.5 % CMC was added and the mixture was stirred until application. Samples were taken to check the amount of radioactive residues in the treatment preparation and to demonstrate the homogeneity and the correctness of the concentration of the test item. In order to confirm the identity and isotope pattern of the test item, an aliquot of the treatment preparation of dose group V was subjected to MS analysis.

Duration and frequency of treatment / exposure:

For balance experiments, the test substance was applied once at concentrations of 3 and 300 mg/kg bw, and repeated oral dosing (by gavage) for 14 days (1 application of 3 mg/kg bw and 14 applications of 300 mg/kg bw/d) was performed with the test substance.
For the tissue distribution experiments, the single treatment was performed at dose levels of 3 and 300 mg/kg bw by gavage.
For bile excretion experiments, the test substance was applied once at dose levels of 3 and 300 mg/kg bw by gavage to bile cannulated rats.

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

oral (gavage)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

oral (gavage)

No. of animals per sex per dose / concentration:

Metabolism study: 4 per sex and dose
Biokinetics study: 4-6 per sex and dose

Control animals:

no

Positive control reference chemical:

no

Details on study design:

Two separate studies have been performed to investigate the biokinetics and the metabolism ofthe test substance. Samples of five dose groups of the biokinetics study were additionally investigated within the metabolism study.

Within the biokinetics study, a series of experiments was carried out with a total of 11 test groups (experiments 1 - 11) to investigate plasmakinetics, bioavailability, distribution and excretion after p.o. (by gavage) or i.v. application of radiolabeledtest substance.

Dose levels for investigation of the plasmakinetics were 3, 30 and 300 mg/kg bw for p.o. administration by gavage and 0.5 mg/kg bw for i.v. administration.

For balance experiments, the test substance was applied once at concentrations of 3 and 300 mg/kg bw, and repeated oral dosing (by gavage) for 14 days was performed with 300 mg/kg bw test substance.

For the tissue distribution experiments, the single treatment was performed at dose levels of 3 and 300 mg/kg bw by gavage.

For bile excretion experiments, the test substance was applied once at dose levels of 3 and 300 mg/kg bw by gavage to bile cannulated rats.

Within the metabolism study, the nature of the biotransformation products ofthe 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. Radiolabeled test substance was applied orally by gavage at dose levels of 3 and 300 mg/kg bw. Plasma, liver, kidney, stomach with contents and carcass samples were generated, whereby plasma, liver and kidney were investigated for isolation and identification of metabolites.

The study design for the metabolism study is shown in Table 1. Liver, kidney and plasma samples were generated during the metabolism study. Radiolabeled urine, feces and bile samples were generated during the biokinetics study. An overview of the origin of all samples is given in Table 1.

Details on dosing and sampling:

Sampling and storage
All samples being generated at the Biokinetics Laboratory, Experimental Toxicology and Ecology, BASF SE were stored in a freezer and transferred to the BASF Agricultural Center, Limburgerhof, in frozen condition. At the Agricultural Center, all samples were stored at -20 °C or below prior to analyses.

The animals of dose groups V and W were anaesthetized with isoflurane and sacrificed by exsanguination 1 h and 4 h, respectively, after administration (time of respective MPC), and samples of liver, kidney and carcass (including stomach with contents) were collected. Blood was collected from the sacrificed animals of dose groups V and W in tubes containing an anti-coagulant and centrifuged to separate plasma from blood cells. Combined plasma samples (several animals per dose group) as well as combined samples of liver, kidney and carcass (including stomach with contents) were stored in a freezer at -20 °C.

In the case of dose group SM, combined daily samples of urine and feces were prepared using equal portions from individual animals.

Equal portions of the bile fluid of several sampling intervals (combined from several animals each) were pooled per sex and dose group to produce samples for the time intervals of 0 – 24 h and 24 – 48 h (dose groups RM, RF, SM and SF) or 0 – 24 h and 24 – 51 h in the case of dose group SF WH.

Determination of radioactive residues
Appropriate aliquots of liquid samples were mixed with a suitable scintillator and measured by LSC. Solid samples were homogenized and appropriate aliquots were combusted using an automated sample oxidizer. The 14CO2 formed during combustion was trapped by an absorption liquid and the collected radioactivity was measured by LSC.

Determination of the total radioactive residue (TRR)
The TRR of urine, bile and plasma was determined by direct LSC. For determination of the TRR of liver and kidney, subsamples were homogenized and combusted. The TRR for feces was calculated as the sum of extracted residues and residual radioactive residues after solvent extraction.

The TRR was used as basis for calculation of the extractability of feces, liver and kidney samples. For dose groups B, D, C, R and S, the referring % dose values were taken from the biokinetics study and used for calculation of metabolite portions in the metabolism study.

HPLC analysis and fractionation
For the determination of the metabolite patterns, metabolite identification, quantitation of metabolites, HPLC fractionation, purity check and stability investigations, several HPLC methods were applied using different reversed-phase columns with gradient elution. For identification and structure elucidation of metabolites, HPLC-MS, HPLC-MS/MS and NMR analyses were performed.

Metabolite analysis by HPLC-MS/MS, NMR
Metabolites of the test substance were identified by high-resolution HPLC-ESI MS and HPLC-MS/MS analysis of the following samples: a urine sample and fractions thereof (dose group SM, 0 – 24 h), feces extracts and fractions of another feces extract (dose group DM, 0 – 24 h; dose group BM, 0 – 24 h) and a bile sample and fractions thereof (dose group SM, 0 – 24 h). Isolated bile fractions (dose group SM) were subjected additionally to NMR analysis. In addition, co-chromatography experiments with reference items (test substance and metabolite M440I017) or samples investigated by HPLC-MS/MS were performed for peak assignment and confirmation.

Extraction and sample preparation for analysis
For analysis of metabolite patterns, pooled urine and bile samples were subjected to radio-HPLC analysis either directly or after dilution with water.
For feces, liver and kidney, homogenized samples were extracted 3x with methanol and 2x with water. After each extraction step, the extract was separated from the solid by centrifugation and filtration, and the solid residue in the centrifugation tube and in the filter was subjected to the next extraction step. The individual extracts (feces from dose group SM, liver and kidney) were adjusted to a defined volume (or the volume was measured), and aliquots were subjected to LSC analysis. Subsequently, the individual methanol and water extracts were combined respectively, concentrated, filtered and appropriate aliquots were subjected to LSC analysis. Samples containing sufficient amounts of radioactive residues were concentrated and subjected to radio-HPLC.

Solid residues of extractions were dried and radioactivity was determined by LSC after combustion. For plasma, samples were purified using acetonitrile for protein precipitation. After addition of acetonitrile, the individual sample was intensely mixed followed by centrifugation and filtration. The residues were washed with acetonitrile, and the supernatants were adjusted to a defined volume and analyzed by LSC and HPLC.

The protein precipitates were suspended in 0.1 M Tris buffer and incubated with protease. The incubation mixtures were centrifuged and filtered, and the combined residues were incubated again with protease. After centrifugation, the supernatants were combined, the volume was measured and the radioactive residues were determined (LSC). The residual precipitates were dried in a fume hood and incubated again with protease. After centrifugation and filtration, the volumes of the supernatants were determined and aliquots analyzed by LSC. The residues in the centrifugation tubes and filters were combined and suspended in water and mixed with tissue solubilizer. After mixing with water and methanol, Hionic-Fluor was added to appropriate aliquots as scintillation cocktail, and the radioactive residues were determined by LSC. The radioactive residues in the protein precipitates were calculated from the first protease solubilizate, the second protease solubilizate and the residues after the second protease treatment.

Details on distribution in tissues:

Distribution of radioactive residues in selected tissues used for the metabolism study
The levels of radioactive residues were investigated in liver, kidney and plasma of male and female rats 1 hour (dose groups VM and VF) and 4 hours (dose groups WM and WF) after dosing with the radiolabelled active substance (Table 2). The highest portions of radioactive residues were recovered in liver for all dose groups ranging from 1.4 % to 7.8 % of the dose. The lowest portions of radioactive residues were measured in plasma ranging from 0.1 % to 0.2 % of the dose.

Details on excretion:

Balance and excretion pattern of 14C labeled test substance
Overall, in all dose groups, excretion was nearly complete within 96 to 120 hours, with more than two thirds of the administered dose being excreted within 48 hours (Table 3). In total, approximately 90 % to 96 % of the dose was excreted via urine and feces over an observation period of about 168 hours. The major part of the radioactive residues was excreted via feces (72 % to 86 % of the dose), while urinary excretion contributed to 5 % to 21 % of the dose.

Metabolites identified:

yes

Details on metabolites:

Metabolite patterns
Metabolites present at 4% or greater of the administered dose were successfully identified. Additional components were not identified, but characterized by a particular molecular mass and the number of cyclopropane carboxylic acid (CPCA) ester moieties using HPLC-MS. In total, the test substance and 26 metabolites (including M440I020 and M440I025) have been identified (see attached document "Proposed metabolic pathway and identified metabolites"). The results per matrix are shown in Table 4 to Table 9 and are summarized in Table 10 and Table 11.

Some components eluted together in one peak and were therefore not quantified individually. For urine, metabolites M440I001 and M440I008 were not separated from their respective glucuronic acid conjugates (M440I054 and M440I043). Further, metabolite M440I002 co-eluted with a characterized component (M = 611 u) and metabolite M440I003 with M440I026. Metabolite M440I019 and metabolite M440I058 were not separated by radio-HPLC and quantified as a sum. However, these two components were separated during HPLC-MS analysis of a purified sub-fraction from bile (dose group SM) and quantified in a ratio of 3.5 : 1 for M440I019 : M440I059 in this bile sample. For feces, metabolite M440I003 was not separated from M440I026. For bile, co-elution of M440I008 and M440I043 as well as M440I019 and M440I059 was observed. Additionally, metabolite M440I001 was not separated from metabolite M440I040 and metabolite M440I035 from metabolite M440I032. According to more detailed data obtained for bile of dose group SM, metabolites M440I001 and M440I035 are proposed to comprise the main constituent of the respective peak also in the other samples.

Investigations of the obtained samples from male and female rats after treatment with the test substance, revealed for urine, ten metabolites and one characterized component still possessing one of the two CPCA ester moieties (in sum approximately 0.8 % – 8.3 % of the dose) and four metabolites, which had lost both CPCA ester moiety (in sum approximately 1.9 % - 11.8 % of the dose). The main biotransformation products excreted via urine were metabolite M440I001 with both CPCA ester moieties lost and its glucuronic acid conjugate M440I054 (not separated and accounting for up to 10.6 % of the dose) and metabolite M440I002 (co eluting with the characterized component with the molecular mass of 611 u; together up to 5.4 % of the dose).

In feces samples, five metabolites had lost one of the CPCA ester moieties (in sum approximately 16.7 % – 32.8 % of the dose) and two metabolites and one characterized component had both CPCA esters cleaved (in sum approximately 21.5 % – 28.2 % of the dose).
The main components found in the extracts from feces of the low dose groups B were the unchanged parent compound (up to 36.7 % of the dose) and the metabolite M440I001 (up to 20.8 % of the dose). In feces from the high dose groups D and C, metabolite M440I001 was the most abundant component (14.9 % – 23.5 % of the dose), followed by metabolite M440I058 (hydroxylated derivative of M440I002; 11.7 % – 15.7 % of the dose). No glucuronic acid conjugates or pyridine N oxidized derivatives were detected in feces.

For bile, eight metabolites and one characterized component had lost one of the CPCA ester moieties (in sum approximately 11.2 % – 20.4 % of the dose) and six metabolites and one characterized component had both CPCA esters cleaved (in sum approximately 2.6% - 6.4% of the dose). The main biotransformation products (up to 28.5 %) detected in bile were metabolite M440I017 (the pyridine N oxidized derivative of the active substance) and M440I019 / M440I059 (not separated).

For the investigated tissues, liver and kidney, and plasma, the main component BAS 440 I was primarily accompanied by metabolites (one to three) with lost CPCA ester moieties, each metabolite accounting for less than 1 % of the dose.

The metabolite patterns were similar for both genders. Some gender differences were observed in detail, e. g. a higher number of minor metabolites in feces extracts of dose groups BM and DM compared to feces extracts of female rats.

In parallel to the kinetics of the total radioactive residues, the metabolites showed the highest % dose values in urine, feces extracts and bile in the first sampling interval and thereafter, values generally decreased with time for both low dose groups. For the high dose groups, most of the metabolites reached their maxima later compared to the low dose groups.

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 dose group D. Only the portion of the N oxidized derivative M440I017 in urine of male rats was lower after administration of the high dose. The relative portions of the parent compound in feces extracts from dose group D were lower, and the portions of metabolites M440I058 and M440I008 were higher compared to extracts from feces of the low dose group B. In bile, the relative portions of the main component M440I017 and of the parent compound were lower in sum, and the portions of most other metabolites were higher for the high dose group S compared to the low group R.

After repeated exposure to the high dose (dose group C), lower relative portions of metabolite M440I002 and slightly higher portions of metabolite M440I039 were observed for instance in urine and feces extracts (dose group C) compared to single treatment with the high dose (dose group D).

Metabolic Pathway
The proposed metabolic pathway of the test substance is shown in the attached document "Proposed metabolic pathway and identified metabolites". An overview on identified metabolites in numerical order is provided as well in the attached document "Proposed metabolic pathway and identified metabolites".

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:
• Hydrolytic loss of one or both CPCA ester moieties (ester cleavage, metabolites M440I001, M440I002 and M440I003)
• N-oxidation at the pyridine-ring (metabolite M440I017 and, in combination with other main reactions, metabolites M440I018, M440I019, M440I027, M440I035 and M440I037)
• Hydroxylation of one of the methyl groups (metabolites M440I034 and M440I026 and, in combination with other main reactions, metabolites M440I008, M440I027, M440I038, M440I039 and M440I058)
• Conjugation of hydroxyl groups of metabolites (M440I001, M440I008 and M440I058) with glucuronic acid (metabolites M440I043, M440I054 and M440I059)

Further metabolites are formed by twofold hydroxylation of the parent molecule at a methyl group and at the pyridine-ring (metabolite M440I032), complete oxidation of the methyl group in position 4 of metabolite M440I002 to a carboxylic acid (metabolite M440I012), dehydrogenation of a hydroxyl group of metabolite M440I039 (metabolites M440I040 and M440I056), dehydration of metabolite M440I019 forming an additional double bond (metabolite M440I036) or reduction of a hydroxyl group of metabolite M440I037 to hydrogen (metabolite M440I028).
In addition, metabolite M440I020, a dehydrogenated derivative of the test substance, was detected as a technical contamination of the application formulation, and metabolite M440I025, a methylated derivative ofthe test substance, was identified in methanol extracts of feces, which has been formed probably as an artefact during sample preparation.

Table 2: Distribution of radioactive residues in plasma and selected tissues at MPC (1 h for dose group V and 4 h for dose group W)

Matrix	Dose group (nominal dose)
	Dose group V 3.0 mg/kg bw				Dose group W 300.0 mg/kg bw
	Male		Female		Male		Female
	% dose	mg/kg1	% dose	mg/kg1	% dose	mg/kg1	% dose	mg/kg1
Liver	7.782	8.031	3.303	3.271	1.357	133.350	1.403	141.470
Kidney	0.534	2.440	0.148	0.760	0.139	66.509	0.148	70.043
Plasma	0.199	0.456	0.096	0.205	0.084	18.226	0.116	27.119

1 TRR measured (calculated using a density of 1.0 g/mL in the case of plasma)

Table 3: Excretion balance (% of administered dose)

Tissue and Interval			3 mg/kg bw								300 mg/kg bw							(14x + 1) 300 mg/kg bw
			BM male			BF 1 female		BF 2 female			DM male			DF female				CM male		CF female
			(cum.)			(cum.)		(cum.)			(cum.)			(cum.)				(cum.)		(cum.)
Urine	0 - 6 h		3.88	3.9		4.70	4.7	4.30	4.3		2.57	2.6		4.27		4.3		4.74	4.7	3.74	3.7
	6 - 12 h		0.80	4.7		0.49	5.2	0.44	4.7		5.34	7.9		6.03		10.3		6.82	11.6	4.97	8.7
	12 - 24 h		0.49	5.2		0.42	5.6	0.45	5.2		7.20	15.1		5.54		15.8		4.93	16.5	4.84	13.6
	24 - 48 h		0.23	5.4		0.17	5.8	0.18	5.4		4.53	19.6		4.02		19.9		0.74	17.2	1.59	15.1
	48 - 72 h		0.04	5.4		0.07	5.9	0.08	5.5		0.23	19.9		1.06		20.9		0.21	17.4	0.14	15.3
	72 - 96 h		0.02	5.5		0.03	5.9	0.03	5.5		0.11	20.0		0.13		21.1		0.12	17.6	0.07	15.4
	96 - 120 h		0.02	5.5		0.02	5.9	0.02	5.5		0.08	20.1		0.08		21.1		0.14	17.7	0.04	15.4
	120 - 144 h		0.01	5.5		0.01	5.9	0.01	5.5		0.07	20.1		0.05		21.2		0.12	17.8	0.03	15.4
	144 - 168 h		0.01	5.5		0.01	5.9	0.01	5.5		0.05	20.2		0.05		21.2		0.09	17.9	0.03	15.5
Subtotal urine			5.49			5.91		5.52			20.19			21.23				17.91		15.44
Feces		0 - 24 h	58.54		58.5	77.45	77.5	70.17	70.2		22.80		22.8		16.89		16.9	32.96	33.0	18.66	18.7
		24 - 48 h	24.73		83.3	7.91	85.4	10.67		80.8	42.15		65.0		32.50		49.4	36.32	69.3	51.68	70.3
		48 - 72 h	2.35		85.6	0.58	85.9	1.52		82.4	6.40		71.4		21.19		70.6	2.72	72.0	7.03	77.4
		72 - 96 h	0.16		85.8	1.43	87.4	1.32		83.7	0.43		71.8		3.18		73.8	0.16	72.2	0.66	78.0
		96 - 120 h	0.05		85.8	0.02	87.4	0.04		83.7	1.12		72.9		0.53		74.3	0.19	72.4	0.09	78.1
		120 - 144 h	0.02		85.9	0.01	87.4	0.05		83.8	0.05		73.0		0.15		74.4	0.04	72.4	0.04	78.2
		144 - 168 h	0.04		85.9	0.02	87.4	0.02		83.8	1.38		74.3		0.05		74.5	0.03	72.4	0.03	78.2
Subtotal feces			85.87			87.43		83.80			74.34				74.49			72.41		78.19
Cage wash			0.06			0.32		0.28			0.15				0.25			0.10		0.12
Blood cells			0.01			0.01		0.01			0.03				0.02			0.02		0.01
Plasma			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Lung			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Heart			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Spleen			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Kidney			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Adrenals			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Testes/Ovaries			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Uterus			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Muscle			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Brain			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Adipose tissue			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Bone			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Bone marrow			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Thyroid			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Pancreas			0.00			0.00		0.00			0.00				0.00			0.01		0.01
Stomach content			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Stomach			0.00			0.00		0.00			0.00				0.00			0.00		0.00
Gut content			0.01			0.01		0.01			0.01				0.01			0.01		0.01
Gut			0.03			0.01		0.01			0.01				0.01			0.01		0.00
Liver			0.01			0.00		0.00			0.02				0.01			0.02		0.02
Skin			0.02			0.02		0.02			0.07				0.04			0.10		0.01
Carcass			0.09			0.12		0.12			0.13				0.13			0.14		0.16
Total recovery			91.60			93.84		89.78			94.96				96.20			90.74		94.00

1 One animal was not included into statistics due to insufficient recovery

2 Calculated with all investigated animals as all samples were used for combining within the metabolism study

Table: 4 Metabolites identified in rat urine (animals treated within the biokinetics study) 

Dose	Percent of administered dose
	Group B Single low dose (3 mg/kg bw)		Group D Single high dose (300 mg/kg bw)		Group C Repeated high dose (14x + 1x 300 mg/kg bw)		Group S Single high dose (300 mg/kg bw)
Compound/Peak 1	Male (0-48 h)	Female (0-48 h)	Male (0-72 h)	Female (0-72 h)	Male (0-48 h)	Female (0-48 h)	Male (0-72 h)
test substance	0.068	0.041	0.058	0.017	0.036	0.116	0.260
M440I001 / M440I054	2.181	1.892	10.551	10.136	9.548	7.806	5.849
M440I002 / M=611 u	0.413	2.151	2.245	5.381	0.964	1.491	2.852
M440I003 / M440I026	0.071	0.131	0.198	0.715	0.303	0.461	0.271
M440I008 / M440I043	0.061	0.024	0.823	0.370	1.310	1.313	0.537
M440I017	0.933	0.438	0.371	0.716	0.688	0.426	0.755
M440I018	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.125
M440I019 / M440I059	0.098	0.142	0.697	0.674	0.227	0.408	1.045
M440I035	0.023	0.004	0.054	0.072	0.104	0.131	0.089
M440I036	n.d.	0.062	0.133	0.256	0.028	0.069	0.109
M440I039	0.313	0.024	0.667	0.312	1.101	0.634	0.524
M440I056	0.174	0.017	0.587	0.099	0.025	n.d.	0.406
M440I058	0.105	0.149	1.689	0.870	0.781	0.926	1.158
Total identified	4.440	5.077	18.072	19.618	15.117	13.781	13.982
M = 539 u	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.051
Sum of components with one (top) or two (bottom) CPCA esters cleaved 2	0.771	2.665	5.838	8.337	3.719	4.799	6.237
	2.668	1.934	11.805	10.547	10.674	8.440	6.780

1 Metabolites are listed in numerical order of the metabolite code

2 For the number of CPCA ester moieties present in the individual components see Table 11

n.d.: not detected/identified

Table: 5 Metabolites identified in rat feces (animals treated within the biokinetics study)

Dose	Percent of administered dose
	Group B Single low dose (3 mg/kg bw)			Group D Single high dose (300 mg/kg bw)		Group C Repeated high dose (14x + 1x 300 mg/kg bw)		Group S Single high dose (300 mg/kg bw)
Compound 1	Male (0-72 h)	Female (0-96 h)	Male 2 (0-168 h)		Female (0-96 h)	Male (0-72 h)	Female (0-72 h)	Male (0-72 h)
test substance	20.688	36.677	10.291		5.317	7.763	6.697	21.223
M440I001	16.898	20.819	17.956		23.496	14.894	21.382	2.784
M440I002	6.088	10.029	9.809		10.125	3.268	4.925	1.591
M440I003 / M440I026	2.629	3.097	0.720		0.320	0.106	1.018	0.293
M440I008	3.387	0.109	7.235		4.793	6.531	7.856	0.095
M440I012	3.592	0.025	1.207		n.d.	3.375	1.005	n.d.
M440I020	0.320	n.d.	n.d.		n.d.	n.d.	n.d.	0.574
M440I025	1.090	n.d.	n.d.		n.d.	n.d.	n.d.	0.117
M440I034	5.415	n.d.	0.106		n.d.	0.500	0.495	n.d.
M440I039	2.501	0.672	3.095		2.254	4.171	4.460	n.d.
M440I058	5.916	3.421	13.213		14.067	11.736	15.658	n.d.
Total identified	68.523	74.849	63.633		60.371	52.345	63.495	26.676
M = 487 u	1.496	0.020	0.314		n.d.	1.558	0.359	n.d.
Sum of components with one (top) or two (bottom) CPCA esters cleaved 3	21.922	16.681	32.194		32.820	25.803	32.011	1.978
	23.702	21.522	23.931		25.775	24.774	28.182	2.784

1 Metabolites are listed in numerical order of the metabolite code

2 Feces sampled within the time intervals 72 – 96 h and 120 – 144 h was not analyzed by HPLC due to low radioactive residues (<1% of the dose)

3 For the number of present CPCA ester moieties see Table 25. Additionally, up to four peaks were characterized by the number of present CPCA ester moieties. The concentrations of these peaks were taken into account for the calculation of the respective sum of components with one or two CPCA esters cleaved

n.d.: not detected/identified

Table 6: Metabolites identified in rat bile (animals treated within the biokinetics study)

Dose	Percent of administered dose
	Group R Single low dose (3 mg/kg bw)		Group S Single high dose (300 mg/kg bw)
Compound 1	Male (0-39 h)	Female (0-39 h)	Male (0-39 h)	Female (0-39 h)
test substance	0.307	1.381	n.d.	0.035
M440I001 / M440I040	3.593	2.252	3.456	3.707
M440I002	0.114	0.320	0.136	0.401
M440I008 / M440I043	2.076	0.337	2.875	2.668
M440I012	0.565	0.876	1.842	1.176
M440I017	20.250	28.518	10.433	8.281
M440I018	1.078	0.158	2.673	1.368
M440I019 / M440I059	6.036	6.687	8.865	6.745
M440I027	n.d.	n.d.	0.223	0.018
M440I028	0.070	n.d.	0.291	0.044
M440I032 / M440I035	1.607	2.221	2.456	2.061
M440I037	0.506	n.d.	0.952	0.716
M440I038	0.409	0.318	1.131	0.559
Total identified	36.610	43.068	35.333	27.779
M = 487 u	0.070	n.d.	0.357	0.197
M = 537 u	0.688	0.558	1.515	2.905
Sum of components with one (top) or two (bottom) CPCA esters cleaved2	12.164	11.157	20.362	17.324
	4.647	2.570	6.409	5.241
1 Metabolites are listed in numerical order of the metabolite code 2 For the number of present CPCA ester moieties in the component see Table 11 n.d.: not detected/identified

Table 7: Metabolites identified in rat liver analyzed at tmax of plasma level (animals treated within the metabolism study)

Dose	Percent of administered dose
	Group V Single low dose (3 mg/kg bw) 1 h after treatment		Group W Single high dose (300 mg/kg bw) 4 h after treatment
Compound 1	Male	Female	Male	Female
test substance	4.685	1.863	0.585	0.673
M440I001 / M440I054	0.438	0.359	0.161	0.104
M440I002	0.928	0.788	0.445	0.410
Total Identified	6.051	3.011	1.191	1.187
Sum of components with one (top) or two (bottom) CPCA esters cleaved2	1.136	0.816	0.504	0.415
	0.438	0.359	0.161	0.104

1 Metabolites are listed in numerical order of the metabolite code

2 For the number of present CPCA ester moieties see Table 11. Additionally, up to two peaks were characterized by the number of present CPCA ester moieties. The concentrations of these peaks were taken into account for the calculation of the respective sum of components with one or two CPCA esters cleaved

Table 8: Metabolites identified in rat kidney analyzed at tmax of plasma level (animals treated within the metabolism study)

Dose	Percent of administered dose
	Group V Single low dose (3 mg/kg bw) 1 h after treatment		Group W Single high dose (300 mg/kg bw) 4 h after treatment
Compound 1	Male	Female	Male	Female
test substance	0.291	0.062	0.085	0.068
M440I001 / M440I054	0.084	0.019	0.020	0.018
M440I002	0.033	0.028	0.019	0.034
Total Identified	0.408	0.109	0.124	0.121
Sum of components with one (top) or two (bottom) CPCA esters cleaved2	0.036	0.028	0.020	0.035
	0.084	0.019	0.020	0.018

1 Metabolites are listed in numerical order of the metabolite code

2 For the number of present CPCA ester moieties see Table 11. Additionally, up to two peaks were characterized by the number of present CPCA ester moieties. The concentrations of these peaks were taken into account for the calculation of the respective sum of components with one or two CPCA esters cleaved

Table 9: Metabolites identified in rat plasma analyzed at tmax of plasma level (animals treated within the metabolism study)

Dose	Percent of administered dose
	Group V Single low dose (3 mg/kg bw) 1 h after treatment		Group W Single high dose (300 mg/kg bw) 4 h after treatment
Compound 1	Male	Female	Male	Female
test substance	0.134	0.032	0.042	0.048
M440I001 / M440I054	0.003	0.005	0.007	0.002
M440I002	0.006	0.014	0.015	0.031
M440I003 / M440I026	0.006	0.005	0.005	0.004
Total Identified	0.150	0.056	0.069	0.085
Sum of components with one (top) or two (bottom) CPCA esters cleaved2	0.012	0.020	0.021	0.035
	0.003	0.005	0.007	0.002

1 Metabolites are listed in numerical order of the metabolite code

2 For the number of present CPCA ester moieties see Table 11. Additionally, up to two peaks were characterized by the number of present CPCA ester moieties. The concentrations of these peaks were taken into account for the calculation of the respective sum of components with one or two CPCA esters cleaved

Table 10: Summary of metabolites identified in urine, feces and bile  

Metabolite (M440I-)

No. of CPCA ester moieties

Urine BM

Urine BF

Urine DM

Urine DF

Urine CM

Urine CF

Urine SM

Feces BM

Feces BF

Feces DM

Feces DF

Feces CM

Feces CF

Feces SM

Bile RM

Bile RF

Bile SM

Bile SF/SF WH

Composition of radioactive residues in % of the dose

000

2

+

+

+

+

+

0.12

0.26

20.69

36.68

10.29

5.32

7.76

6.70

21.22

0.31

1.38

n.d.

+

001

0

2.18^3

1.89^3

10.55^3

10.14^3

9.55^3

7.81^3

5.85^3

16.90

20.82

17.96

23.50

14.89

21.38

2.78

3.59^6

2.25

3.46

3.71

002

1

0.41^4

2.15^4

2.24^4

5.38^4

0.96^4

1.49^4

2.85^4

6.09

10.03

9.81

10.13

3.27

4.93

1.59

0.11

0.32

0.14

0.40

003 / 026

1 / 2

+

0.13

0.20

0.72

0.30

0.46

0.27

2.63

3.10

0.72

0.32

0.11

1.02

0.29

n.d.

n.d.

n.d.

n.d.

008

1

+5

+5

0.82^5

0.37^5

1.31^5

1.31^5

0.54^5

3.39

0.11

7.24

4.79

6.53

7.86

+

2.08^5

0.34

2.87

2.67

012

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

3.59

+

1.21

n.d.

3.37

1.00

n.d.

0.57

0.88

1.84

1.18

017

2

0.93

0.44

0.37

0.72

0.69

0.43

0.76

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

20.25

28.52

10.43

8.28

018

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.13

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.08

0.16

2.67

1.37

019 / 059

1 / 1

+

0.14

0.70

0.67

0.23

0.41

1.05

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

6.04

6.69

8.87

6.74

0201

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.32

n.d.

n.d.

n.d.

n.d.

n.d.

0.57

n.d.

n.d.

n.d.

n.d.

0252

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.09

n.d.

n.d.

n.d.

n.d.

n.d.

0.12

n.d.

n.d.

n.d.

n.d.

027

0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.22

+

028

0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

+

n.d.

0.29

+

032 / 035

2 /1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.61

2.22

2.46

2.06

034

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

5.41

n.d.

0.11

n.d.

0.50

0.50

n.d.

n.d.

n.d.

n.d.

n.d.

035

1

+

+

+

+

+

0.13

+

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

036

1

n.d.

+

0.13

0.26

+

+

0.11

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

037

0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.51

n.d.

0.95

0.72

038

0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.41

0.32

1.13

0.56

039

0

0.31

+

0.67

0.31

1.10

0.63

0.52

2.50

0.67

3.10

2.25

4.17

4.46

n.d.

n.d.

n.d.

n.d.

n.d.

056

0

0.17

+

0.59

+

+

n.d.

0.41

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

058

1

0.11

0.15

1.69

0.87

0.78

0.93

1.16

5.92

3.42

13.21

14.07

11.74

15.66

n.d.

n.d.

n.d.

n.d.

n.d.

M=487

0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.50

+

0.31

n.d.

1.56

0.36

n.d.

+

n.d.

0.36

0.20

M=537

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

+

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

M=539

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.69

0.56

1.52

2.91

CPCA Cyclopropane carboxylic acid

+ Metabolite was assigned, but the % of the dose value was equal or below 0.1%.

n.d. Not detected

1 Impurity

2 Artefact

3 Includes metabolite M750F054 (no CPCA ester mojety)

4 Includes a metabolite with molecular mass M = 611 (two CPCA ester mojeties)

5 Includes metabolite M750F043 (one CPCA ester mojety)

6 Includes metabolite M750F040 (no CPCA ester mojety)

Table 11: Summary of metabolites in tissues and plasma

Metabolite(M440I-)	No of CPCA ester moieties	Liver				Kidney				Plasma
		VM	VF	WM	WF	VM	VF	WM	WF	VM		VF		WM		WF
		Composition of Radioactive Residues in % of the Dose
000	2	4.68	1.86	0.58	0.67	0.29	0.06	0.09	0.07	0.13	0.03		0.04		0.05
001 / 054	0 / 0	0.44	0.36	0.16	0.10	0.08	0.02	0.02	0.02	+	+		+		+
002	1	0.93	0.79	0.45	0.41	0.03	0.03	0.02	0.03	+	+		0.02		0.03
003 / 026	1 / 2	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	+	+		+		+

+ Metabolite was assigned, but the % of the dose value was equal or below 0.01%.

n.d. not detected/identified

Extractability and analysis

For the investigation of the extractability of the radioactive residues as well as the metabolite patterns in feces, liver and kidney, samples were extracted three times with methanol and twice with water. No gender-related differences in the extraction pattern were observed.

The extractability of radioactive residues in feces of male and female rats of dose groups B, D, C and S (male only) was very high ranging from 85% to 97% of the total radioactive residues (TRR). Major portions of radioactive residues were extracted with methanol, and only minor portions (up to 4.1% TRR) were subsequently extracted with water. Due to the low residue levels (below or equal to 10% TRR or below 1% of the dose), the residual radioactive residues after extraction with methanol and water were not further investigated.

The radioactive residues of the test substance in liver of rats of dose groups V and W were very effectively extracted with methanol and water (94% to 100% TRR). Portions of 98% to 101% TRR were extracted in total from kidney of rats of dose groups V and W. In any case, major portions of the radioactive residues were extracted with methanol, the subsequent extraction with water released only minor portions below 1.5% TRR. There was no need for further investigations of the residual radioactive residues after solvent extraction (below 1% TRR in each case).

Reference 7

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 2014-07-07 To: 2015-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 28 for the conduct of skin absorption studies

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABLELED TEST MATERIAL
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1055-0201

RADIOLABELLING INFORMATION
- Radiochemical purity: > 95 %
- Specific activity of AI: 3.7 MBq/mg
- Specific activity: 16.7 MBq/g test substance solution
- Locations of the label: pyranone-6-C14

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: freezer
- Homogeneity: given

FORMULATION CONCENTRATE
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: FD-140131-0011
- Composition: Formulation; active substance: nominal content : 50.0 g/L; analyzed content : 48.2 g/L
- Density: 1.025 g/cm3

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Homogeneity: given
- Storage stability: expiry date: 2016-02-27
- Physical state / appearance: liquid/yellowish, clear

BLANK FORMULATION (WITHOUT TEST SUBSTANCE)
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: FD-140624-0028
- Density: 1.027 g/cm³

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Homogeneity: given
- Storage stability: expiry date: 2016-02-27

NON-LABELED TEST MATERIAL
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: COD-001545
- Purity: 97.3 % (tolerance +/-1.0 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, hygroscopic; keep away from humidity
- Homogeneity: given
- Storage stability: expiry date: 2016-01-31
- Physical state / appearance: solid/yellowish

STABILITY, HOMOGENEITY AND CONCENTRATION CONTROL ANALYSES OF THE TEST-SUBSTANCE PREPARATIONS
Stability of the testsubstance preparation: The stability of the test substance in the test-substance preparations over the test period was verified by analyses.
Concentration control and homogeneity analysis of the test-substance preparation: The concentration and the homogeneous distribution of the test substance in the test-substance preparations were confirmed by analyses.

See also Table 1 and 2.

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl: WI (Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 97633 Sulzfeld, Germany
- Age at study initiation: about 10 weeks
- Weight at study initiation: about 290–365 g prior to dosing
- Housing: During acclimatization animals housed in groups (up to five animals) in Polysulfonate cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2); during the experiment they were kept individually in all-glass metabolism cages type Metabowl (Jencons Leighton Buzzard, U.K.), labeled with the project number, animal number, dose and time of application.
- Diet: Kliba lab diet (mouse / rat “GLP”) meal. Origin:Provimi Kliba SA, 4303 Kaiseraugst, Switzerland ad libitum prior to and during the experiment
- Water: tap water ad libitum
- Acclimation period: at least 8 days before the beginning of the experimental phase; during the acclimatization period, the animals were accustomed to the environmental conditions of the study and to the diet

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 – 70 %
- Air changes: 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2014-07-07 To: 2014-12-04

Type of coverage:

semiocclusive

Remarks:

To protect the test area, a glass saddle was placed around the spacer and the whole application site was covered with a permeable (gauze) dressing and a semi occlusive adhesive bandage above

Vehicle:

other: Pure radiolabeled test substance was taken and was added to the blank formulation mixed with tap water at a ratio of 1:100 (v:v) and 1:200 (v:v) for the mid and low dose, respectively.

Duration of exposure:

Group A B C
Duration of exposure [h] 8 8 8
Skin wash after [h] 8 8+24 8+120
Skin strip after [h] 8 24 120
Sacrifice after [h] 8 24 120
Number of animals 4 4 4

Doses:

14C-test substance in the formulation concentrate was applied at three concentrations.

For the calculation of the dermal dose, it was considered that 100 μL of the test-substance preparation was applied to a total area of about 10 cm².

Dermal application was selected in order to simulate user specific exposure scenarios. According to the relevant guidelines, experiments shall be performed using doses expected in field exposure. Here, doses were selected to cover a broad range of potential exposure during use.

See also Table 3.

No. of animals per group:

4 animals/group

Control animals:

no

Details on study design:

TEST-SUBSTANCE PREPARATIONS
High dose:
To obtain the desired specific activity, a respective aliquot of the solution of the radiolabeled test substance was taken and the organic solvent (acetonitrile) was evaporated to dryness. An appropriate amount of the formulation concentrate was added to the dried residue. Based on the current data, the nominal concentration of the test substance in the formulation concentrate was 49.5 mg/g (corresponding to 50.8 mg/mL). The nominal specific activity in the test substance preparation was 9.8 MBq/g (corresponding to 10.0 MBq/mL). The applied mean radioactive dose per animal was 0.97 MBq.

Mid dose and low dose:
Due to the low concentration of active ingredient in the 1:100 (v:v) and 1:200 (v:v) aqueous spray dilutions, pure radiolabeled test substance was taken and was added to the blank formulation mixed with tap water at a ratio of 1:100 (v:v) and 1:200 (v:v) for the mid and low dose, respectively. Therefore appropriate amounts of the stock solution of the radiolabeled test substance were evaporated to dryness and then mixed with an appropriate amount of the previously prepared blank formulation mixed with tap water. The results of the measured radioactivity of the first run of the mid dose showed a declined amount of radioactivity in the spray dilution 1 with correlated inhomogeneity between samples of the test-substance preparation taken before and after application. Aditionally, recoveries were insufficient recoveries for analysed samples of this dose group. Therefore this experiment was repeated and the first preparation is not outlined in the report in detail. Based on the assumption that the inhomogeneity and incompete recovery was caused by absorption of the test substance to the glass surface, the surface of the glassware was inertisized by pretreatment with Dichlorodimethylsilane before it was used for the test-substance preparations of the low dose and the repetition of the mid dose. Since only radiolabeled test substance was used for both concentrations the amount of test substance was calculated by the measured amount of activity in the spray dilutions.

Based on the current data, the measured concentrations of the test substance in the formulation concentrate preparations were 0.48 mg/g (corresponding to 0.48 mg/mL) for the mid dose and 0.27 mg/g (corresponding to 0.27 g/mL) for the low dose. The measured specific activities in the test-substance preparations were 1.78 MBq/g (corresponding to 1.78 MBq/mL) and 1.0 MBq/g (corresponding to 1.0 MBq/mL) for the mid and the low dose, respectively.

The applied mean radioactive dose per animal was 0.2 MBq for the mid dose and 0.1 MBq for the low dose.

All preparations were stirred, the preparations of the mid and low dose additionally ultrasonicated in order to produce homogeneous preparation. At least before start and at the end of the application, samples were taken to determine the amount of radioactivity in the preparations and to demonstrate the correct concentrations of the test substance, its homogeneity and its radiochemical purity.

ANALYSES/MEASUREMENTS
Homogeneity/Concentration control and stability analyses
The amounts of radioactivity of 14C-test substance in the formulation concentrate preparations were determined in samples that were taken at least before and after the application at the laboratory for Biokinetics. The analyses of these samples allow to demonstrate the homogeneity, correctness of the concentration and the stability of the test substance in the test-substance preparation.

Stability in vehicle: The stability in the vehicle was investigated by Radio-HPLC and HPLC/UV.
Homogeneity and correctness of the concentrations in the vehicle: The homogeneity and correctness of the concentrations were verified by LSC and HPLC.

Methods of analysis
The analyses of the test-substance preparations were performed by HPLC (HP 1100 system and HP 1300 system) according to the following conditions:
Column: Luna C18, 250 mm x 3 mm
Eluent: A: HPLC water
B: acetonitrile
Flow: 0.5 mL/min, 50 % B, isocratic
Detection: UV-extinction 230 nm HPLC Radioactivity Monitor LB 509 (cell: YG-75)

Weight
The body weight was determined on the day of application prior to dosing. The weights of all samples were determined, for skin wash samples, application material and tape strips, weights were determined with the added amount of Soluene®-350 (Perkin Elmer).

Total radioactivity in biological material and samples used for washing and protecting the application site
Analyses and measurements were performed according to following work up procedures:
After weighing, aliquots of liquid samples (urine, plasma and cage wash) were mixed with scintillation cocktail (Hionic Fluor, Perkin Elmer) and analyzed for radioactivity without any additional treatment.
Soluene®-350 (Perkin Elmer) was added to blood cells. The samples were incubated at room temperature and isopropanol was added afterwards. Then the samples were bleached with perhydrol solution (30%). After further incubation Hionic Fluor cocktail (Perkin Elmer) was added and the samples were measured by LSC.
The samples of terminal skin wash (cotton swabs with washing solution), skin wash (cotton swabs with washing solution), application material after exposure (glass saddle, rubber ring and bandage), and after post-observation period (bandage) as well as tape strips were placed in appropriate containers and Soluene®-350 (Perkin Elmer) was added (100-200 g to the application material after exposure, 50-100 g to the application material after post-observation, 10-20 g to the tape strips, 15-30 g to the terminal skin wash and 40-60 g to the skin wash after exposure). The samples were extracted with Soluene®-350 (Perkin Elmer) for several days at room temperature. Then scintillation cocktail (Hionic Fluor, Perkin Elmer) was added to aliquots of the extracts and the samples were counted in a LSC.
Feces and carcass were suspended in ultrapure water and were homogenized using a WARING Blender. Aliquots of the suspensions were dried by lyophylisation, dissolved in Soluene®-350, filled up with isopropanol, bleached with perhydrol solution (30%) and were incubated afterwards. Hionic Fluor was added and the samples were measured by LSC.
Skin from the application site and surrounding skin was cut into three pieces each. The pieces were placed into scintillation vials and Soluene®-350 (Perkin Elmer) was added (3 to 4 mL). The samples were incubated and isopropanol was added afterwards. Then, bleaching was performed with perhydrol solution (30%) followed by incubation. Hionic Fluor cocktail (Perkin Elmer) was added and the samples were measured by LSC.

EXPERIMENTAL PROCEDURE
Application of the test material 24 hours before treatment, the back of the animals was thoroughly clipped and then the application site was washed with acetone. On the day of dosing, a spacer was glued to the skin with tissue glue. The test substance preparation was applied to the application site (dosing volume: 10 μL/cm²; treated area: about 10 cm²). The pipette used was weighed before and after application. To protect the test area, a glass saddle was placed around the spacer and the whole application site was covered with a permeable (gauze) dressing and a semi occlusive adhesive bandage above.

Balance/excretion
After treatment, animals were placed in metabolism cages. Excreta was collected over the respective complete exposure period (8 h) and – where appropriate – additionally after 24 and at intervals of 24 hours up to a maximum of 120 h.

Washing procedure:
After the 8 hour exposure period, the gauze and bandages as well as the rubber ring and the glas saddle were removed from the animals. The skin of the animals was washed with a mild soap solution [Texapon ® N 70 (Sodium-laurylethersulfate, Cognis, Germany)] and rinsed with water.
The washing procedure was:
- Cotton swabs dampened with water and liquid soap
- Area inside the glass saddle washed using a circular motion
- Cotton swabs dampened with water
- Area inside the glass saddle wiped using a circular motion
- The wiping was repeated several times with new swabs and finally the area was dried with at least one dry cotton swab
For animals with a post-observation period, a new gauze and bandage was applied after the 8 h skin wash and an additional skin wash (according to the 8 hours skin wash) was performed before sacrifice. The washing solutions and the gauze with bandage were collected separatly from those at the end of exposure for determination of residual radioactivity.

Tape stripping:
Scotch tape strips (stripping was performed once with two tape strips in parallel for the high dose (to strip the complete application area) and twice with two tape strips in parallel for the mid and low dose) were taken from the application site after the final surface wash to check for further easily removable test material. The tape strips were pooled and analyzed.

Mortality
A check for moribund and dead animals was conducted at least once daily.

Clinical signs
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

Signs and symptoms of toxicity:

no effects

Dermal irritation:

not specified

Absorption in different matrices:

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.

High dose:
At all sampling time points, the largest proportion of radioactivity was recovered from the skin wash. The mean radioactive recoveries in skin wash was 88.72% (sacrifice after 8 h), 82.72% (sacrifice after 24 h) and 81.54% (sacrifice after 120 h), respectively. Second skin washes contained 1.17 and 0.15% of the administered radioactivity for the dose groups being sacrificed after 24 and 120 h, respectively.
Mean radioactivity in the protective cover of the exposure period ranged from 1.42 – 9.61% of the radioactivity applied. Mean radioactivity in the protective cover of the post exposure periods were 4.88 (24 h) and 6.00% (120 h) of the radioactivity applied. In the animals sacrificed directly after the 8 h exposure period, the application site and the surrounding skin contained means of 2.07 and 0.82% of the applied radioactivity. For the animals with post-observation periods of 24 h and 120 h, the mean radioactivity at the application site amounted to 1.26% and 0.48% of the radioactivity applied, respectively. The amounts in the skin surrounding the application site were 0.76 and 0.27% of the radioactivity applied. Skin strips, performed with scotch tapes after sacrifice accounted for means of 0.25%, 0.07% and 0.03% of the applied radioactivity for the animals of the 8 h, 24 h and 120 h sacrifice group.
The mean amounts of radioactivity absorbed (including excreta, cage wash, carcass, blood cells and plasma) were 0.37%, 0.69% and 1.22% of the applied dose at 8 h, 24 h and 120 h after beginning of the 8 h exposure period, respectively. These results together with the amounts in the skin (application site and surrounding skin) and the values of the second skin wash imply that at this dose level less than 30% of the radioactivity remaining with the skin after end of exposure penetrates through the skin during the post-observation period. The radioactivity absorbed was excreted via urine and faeces with higher amounts in faeces at later time points. The highest tissue concentrations of radioactivity were found in carcass for the dose groups being sacrificed after 8 and 24 hours and in blood cells for the dose group being sacrificed after 120 hours. Individual samples of blood cells with activities below background (that was unusual high, with a value of 108.5 cpm) yielded a tissue concentration of formally 0.00 μg Eq/g. Correspondingly, this fact leads (for individual samples) to artificial low blood/plasma ratios. However, since the absolute values of background and the residues in these samples are low, the observed background variability is negligible and have no impact on the overall data assessment of the study.

Mid dose:
At all sampling time points, the largest proportion of radioactivity was recovered from the skin wash. The mean radioactive recoveries in skin wash were 79.47% (sacrifice after 8 h), 78.43% (sacrifice after 24 h) and 78.63% (sacrifice after 120 h), respectively. Second skin washes contained 2.18 and 0.97% of the administered radioactivity for the dose groups being sacrificed after 24 and 120 h, respectively.
Radioactivity in the protective cover of the exposure period ranged from 3.87 to 5.87% of the radioactivity applied. Mean radioactivity in the protective cover of the post exposure periods were 1.09 (24 h) and 3.99% of the radioactivity applied (120 h). In the animals sacrificed directly after the 8 h exposure period, the application site and the surrounding skin contained means of 9.74 and 0.12% of the applied radioactivity. For the animals with post-observation periods of 24 h and 120 h, the radioactivity at the application site amounted to 8.74% and 5.52% of the radioactivity applied, respectively. The mean amounts in the skin surrounding the application site were 0.10 (120 h) 0.17% of the radioactivity applied (24 h). Skin strips, performed with scotch tapes after sacrifice accounted for means of 0.23%, 0.16% and 0.19% of the applied radioactivity for the animals in the 8 h, 24 h and 120 h sacrifice group.
The mean amounts of radioactivity absorbed (including excreta, cage wash, carcass, blood cells and plasma) were 2.89%, 1.87% and 3.48% at 8 h, 24h and 120 h after beginning of the 8 h exposure period, respectively. These results together with the amounts in the skin (application site and surrounding skin) and the values of the second skin wash imply that at this dose level less than 15% of the radioactivity remaining on the skin after end of exposure penetrates through the skin during the post-observation period. The radioactivity absorbed was excreted via urine and feces with higher amounts in feces at later time points. The highest tissue concentrations of radioactivity were found the in blood cells.


Low dose:
At all sampling time points, the largest proportion of radioactivity was recovered from the skin wash. The mean radioactive recoveries in skin wash were 80.07% (sacrifice after 8 h), 73.49% (sacrifice after 24 h) and 74.81% (sacrifice after 120 h). Second skin washes contained means of 2.14 and 1.06% of the administered radioactivity for the dose groups being sacrificed after 24 and 120 h, respectively.
Mean radioactivity in the protective cover of the exposure period ranged from 1.38 to 5.43% of the radioactivity applied. Mean radioactivity in the protective cover of the post exposure period was 1.75 (24 h) and 4.49% of the radioactivity applied (120 h). In the animals sacrificed directly after the 8 h exposure period, the application site and the surrounding skin contained means of 10.45 and 0.12% of the applied radioactivity. For the animals with post-observation periods of 24 h and 120 h, the mean radioactivity at the application site amounted to 7.29% and 5.73% of the radioactivity applied, respectively. The mean amounts in the skin surrounding the application site were 0.07 (120 h) and 0.09% of the radioactivity applied (24 h). Skin strips, performed with scotch tapes after sacrifice accounted for means of 0.43%, 0.15% and 0.16% of the applied radioactivity for the animals in the 8 h, 24 h and 120 h sacrifice group.
The mean amounts of radioactivity absorbed (including excreta, cage wash, carcass, blood cells and plasma) were 4.25%, 4.67% and 5.55% at 8 h, 24 h and 120 h after beginning of the 8 h exposure period, respectively. These results together with the amounts in the skin (application site and surrounding skin) and the values of the second skin wash imply that at this dose level less than 15% of the radioactivity remaining on the skin after end of exposure penetrated through the skin during the post-observation period. The radioactivity absorbed was excreted via urine and feces with higher amounts in feces at later time points. The highest tissue concentrations of radioactivity were found in the blood cells.


See also Table 1 and Table 2.

Total recovery:

- Total recovery: 93.25 - 98.83 % (2.5 µg/cm²); 96.31 - 98.52 % (5.0 µg/cm²); 92.96 - 99.30 % (500 µg/cm²)
- Recovery of applied dose acceptable: Yes

Time point:

8 h

Dose:

500 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

0.37 %

Time point:

24 h

Dose:

500 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

0.69 %

Key result

Time point:

120 h

Dose:

500 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

1.22 %

Time point:

8 h

Dose:

5.0 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

2.89 %

Time point:

24 h

Dose:

5.0 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

1.87 %

Key result

Time point:

120 h

Dose:

5.0 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

3.48 %

Time point:

8 h

Dose:

2.5 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

4.25 %

Time point:

24 h

Dose:

2.5 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

4.67 %

Key result

Time point:

120 h

Dose:

2.5 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

5.55 %

Conversion factor human vs. animal skin:

not applicable

Table 1: The mean percentages of absorbed dose

mean percentage and ± SD of                       ^14 C-test substance in 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

Table 2: Mean excretion and retention of radioactivity after a single dermal application of ¹⁴C-BAS 440 I in BAS 440 01 I to rats at nominal dose levels of 500, 5.0 and 2.5 μg/cm ². Results expressed in % of dose administered

Target dose [μg/cm²]	500	500	500	5.0	5.0	5.0	2.5	2.5	2.5
Exposure time [h]	8	8	8	8	8	8	8	8	8
Sacrife time [h]	8	24	120	8	24	120	8	24	120
Dose admin. [mg/cm²]	0.49	0.49	0.49	0.0048	0.0048	0.0048	0.0026	0.0026	0.0026
Urine -8	0.02	0.02	0.02	0.11	0.07	0.12	0.15	0.15	0.17
Urine -24	---	0.05	0.04	---	0.07	0.09	---	0.12	0.18
Urine -48	---	---	0.03	---	---	0.05	---	---	0.06
Urine -72	---	---	0.02	---	---	0.02	---	---	0.04
Urine -96	---	---	0.01	---	---	0.01	---	---	0.02
Urine -120	---	---	0.01	---	---	0.01	---	---	0.02
Subtotal Urine	0.02	0.06	0.13	0.11	0.14	0.30	0.15	0.27	0.48
Feces -8	0.00	0.00	0.01	0.03	0.02	0.01	0.02	0.03	0.03
Feces -24	---	0.25	0.17	---	0.55	0.62	---	0.74	0.57
Feces -48	---	---	0.25	---	---	0.85	---	---	0.94
Feces -72	---	---	0.16	---	---	0.36	---	---	0.46
Feces -96	---	---	0.15	---	---	0.34	---	---	0.21
Feces -120	---	---	0.07	---	---	0.18	---	---	0.09
Subtotal Feces	0.00	0.25	0.81	0.03	0.57	2.34	0.02	0.77	2.29
Cage wash	0.00	0.01	0.03	0.00	0.00	0.11	0.00	0.00	0.00
Blood cells	0.00	0.00	0.01	0.08	0.05	0.05	0.13	0.08	0.06
Plasma	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Carcass	0.35	0.36	0.25	2.67	1.12	0.68	3.95	3.56	2.72
Percentage absorbed	0.37	0.69	1.22	2.89	1.87	3.48	4.25	4.67	5.55
Surrounding skin	0.82	0.76	0.27	0.12	0.17	0.10	0.12	0.09	0.07
Protective cover expos.	4.44	1.42	9.61	3.87	5.87	5.16	3.52	5.43	1.38
Protective cover post expos.	n.s.	4.88	6.00	n.s.	1.09	3.99	n.s.	1.75	4.49
Application site	2.07	1.26	0.48	9.74	8.74	5.52	10.45	7.29	5.73
Skin wash	88.72	82.72	81.54	79.47	78.43	78.63	80.07	73.49	74.81
2nd skin wash	n.s.	1.17	0.15	n.s.	2.18	0.97	n.s.	2.14	1.06
Skin strip	0.25	0.07	0.03	0.23	0.16	0.19	0.43	0.15	0.16
Total	96.65	92.96	99.30	96.31	98.52	98.02	98.83	94.99	93.25

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 AUC_{0 -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 T_max 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: Pharmacokinetics 2019/2075281

The objective of this study was to evaluate the pharmacokinetics of BAS 440 I (Afidopyropen) and of its three metabolites M440I017 (BASF Reg. No. 6045738), M440I001 (BASF Reg. No. 5741530), and M440I060 (BASF Reg. No. 6009307) in rats. F344 rats (F344/DuCrlCrlj) of both sexes were fed diets containing the test substance at concentrations adjusted to equate the target dose levels of 0.1, 0.5, 2.5, 12.5, 50, and 150 mg/kg/day every week for a period of 28 days (4 weeks). Each dose group consisted of 6 animals per sex. Pharmacokinetic analyses were conducted for both sexes based on blood samplings at days 21 and 28 and the concentrations of BAS 440 I (Afidopyropen) and the three metabolites M440I017 (BASF Reg. No. 6045738), M440I001 (BASF Reg. No. 5741530), and M440I060 (BASF Reg. No. 6009307) were quantified in each individual plasma sample. Under the chosen test conditions, it could be demonstrated in principle that BAS 440 I (Afidopyropen) and the metabolites M440I001 and M440I060 show a linear kinetics at low doses tested but switch to a disproportionate increase in internal doses over external doses when the external dose level exceeds 0.5 mg/kg/day.

 

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.