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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2017

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2020

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD 414, Commission Regulation (EC) No 440/2008 - Part B No. B.31, EPA 870.3700

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate

EC Number:

815-966-6

Cas Number:

915972-17-7

Molecular formula:

C33H39NO9

IUPAC Name:

[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate

Test material form:

solid

Details on test material:

- Analytical purity: 96.1 %
- Appearance: Solid yellowish
- Batch: COD-001545

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- identification: test substance (206477)
- batch number of test material: COD-001545
- Appearance: yellow solid
- Purity: 96.15%

Analytical standards:
- Identification: AS1574, CPCA-carnitine (M440I060, 15/0440-1)
- Appearance: white powder
- Batch: L82-166
- Purity: 97.8% (tolerance +-1.0%)

- Identification: AS1563, Reg.No. 6045738 (Metabolite of Afidopyropen) (M440I017, 15/0199-2)
- Appearance: yellowish solid
- Batch: L82-178
- Purity: 94.8% (tolerance +- 1.0%)
- Purity/composition correction factor: correction factor 1.067 for purity

- Identification: AS1561, Reg. No. 5741530 (Metabolite of Afidopyropen, M440I001, 15/0196-1)
- Appearance: yellowish solid
- Batch: L82-66
- Purity: 93.9% (tolerance +-1%)
- Correction factor for purtity: 1.065

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature

Radiolabelling:

no

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-19 weeks
- Weight at study initiation: on day 6 post-coitum, start of dosing: 3.2-4.1 kg
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): >10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: 09 November 2016 To: 25 November 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous CMC

Duration and frequency of treatment / exposure:

single and repeated oral gavage administration(s);
- Once daily for 7 days per week;
- Exposure period: From day 6 to day 13 post-coitum (incl)
- Dose volume: 2 mL/kg bw

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

32 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 animals per dose level (16 rabbits)

Details on study design:

This study investigates the toxicokinetics of the test substance in pregnant rabbits after single and repeated oral gavage administration(s).
The toxicokinetic plasma profile of the test substance and its metabolites M440I001, M440I017 and CPCA-carnitine were assessed in healthy pregnant female rabbits after single and repeated oral administration at doses of 5, 15, 32 and 60 mg BAS 440 I/kg bw for 8 days.

Details on dosing and sampling:

Dose administration:
The test substance was administered once daily for 7 days/week from day 6 to day 13 post-coitum via gavage at a volume of 2mL/kg bw each.

Pharmacokinetic Blood Sampling
On Days 6 and 13 post-coitum, blood was collected from the ear artery. Animals were not fasted prior to sampling. The ear artery was punctured with a needle and blood samples of approximately 0.3 mL blood samples were collected directly into tubes containing K2-EDTA as anti-coagulant. In the case a lesion on the ear (caused by earlier blood sampling) could be opened to collect a blood sample, it was not necessary to use a needle. Blood was collected from all rabbits at the following time points:
Day 6: predose, 0.5 (30 min), 1, 2, 4, 8 and 24 hours after dosing
Day 13: predose, 0.5 (30 min), 1, 2, 4, 8, 24, 48 and 72 hours after dosing
All blood samples were placed on melting ice immediately after sampling, blood was centrifuged within 1 hour after sampling at approximately 3220g for 10 minutes at 4-8 °C. Immediately after centrifugation, plasma was stored in labeled polypropylene tubes at ≤ -75°C until analysis.

Bioanalysis
Study samples were analyzed according to the fit for purpose bioanalytical method. The study samples were analyzed for their content of BAS 440 I (206477), M440I001 (AS1561), M440I017 (AS1563) and CPCA-carnitine (AS1574).

The samples were directly analyzed or stored in an ultra- low freezer (≤ -75°C) until analysis. Any remaining samples were returned to storage for the retention period.

Pharmacokinetic Evaluation
If the test item and metabolites were detectable in plasma, all pharmacokinetic parameters were calculated from the curves from individual animals using the validated Phoenix WinNonlin 6.3 program. Non-compartmental analysis was applied, using the extravascular model. Nominal sampling times were used in pharmacokinetic calculations. Nominal dose levels were used except for the low dose group (Group 1, 5 mg/kg) on Day 13 post-coitum where the actual dose level (4.18 mg/kg) was used based on the formulation analysis. Non- pregnant animals were excluded from the PK evaluation.

Calculated parameters:

- Cmax: Maximum plasma concentration based on bioanalytical results, i.e. not estimated based on curve-fitting.
- Clast: Last measurable plasma concentration.
- tmax: Time point at which maximum plasma concentration was reached, assessed directly from the data.
- tlast: Time point of last measurable plasma concentration.
- AUClast: Area under the plasma concentration-time curve from time of administration until the last measurable plasma concentration (tlast), calculated using the linear-logarithmic trapezoidal rule. A partial area (AUC0-24) was calculated on Day 13 post-coitum.
- AUC∞: Area under the curve after a single dose from time of administration until infinity, calculated as AUClast + Clast/λz, where Clast was the last measurable concentration. If the data set did not allow extrapolation to infinity, then the AUC up to the last measurable time point was calculated. In case the extrapolation to infinity was more than 20% of the total AUC, the AUC∞ was not reported. When t1/2 was not reported, AUC∞ was also not reported.
- λz: Elimination rate constant, determined by linear regression of the terminal points of the ln-linear concentration-time curve.
- t½: Elimination half-life, calculated as ln(2)/λz. The following requirements had to be met for an acceptable calculation of t½:
1) at least three time points were available was used in the calculation
2) correlation coefficient (r2) was at least 0.8
3) span of time points used in t½ was at least twice the calculated value of t½
Values that did not meet these criteria were not reported.

Results and discussion

Any other information on results incl. tables

Parameters		Group 1 (5 mg/kg)	Group 2 (15 mg/kg)	Group 3 (32 mg/kg)	Group 4 (60 mg/kg)
test substance
Day 6
tlast	(h)	2-4$	4-8$	8-24$	24
tmax	(h)	0.5	0.5-1$	0.5-1$	1-2$
Cmax	(ng/mL)	15.6	438	2780	4810
AUClast	(h∙ng/mL)	12.6	600	6110	24100
AUC∞	(h∙ng/mL)	n/a	500	n/a	24100
t½	(h)	n/a	0.961	n/a	1.99
Day 13		(4.18 mg/kg)
tlast	(h)	2-8$	8-24$	8-72$	72
tmax	(h)	0.5-2$	0.5-2$	0.5-4$	1-4$
Cmax	(ng/mL)	9.57	447	2490	5260
AUClast	(h∙ng/mL)	13.6	635	7350	101000
t½	(h)	n/a	n/a	1.60	4.95
M440I001
Day 6
tlast	(h)	24	24	24	24
tmax	(h)	0.5-2$	1-2$	1	1-2$
Cmax	(ng/mL)	66.8	185	898	1150
AUClast	(h∙ng/mL)	207	730	4670	8590
AUC∞	(h∙ng/mL)	231	870	4910	8800
t½	(h)	6.36	4.41	4.72	4.21
Day 13		(4.18 mg/kg)
tlast	(h)	24-72$	48-72$	72	72
tmax	(h)	0.5-2$	0.5-2$	1-4$	1-4$
Cmax	(ng/mL)	55.5	193	837	1170
AUClast	(h∙ng/mL)	302	1090	6600	28700
t½	(h)	n/a	11.6	15.3	11.1
M440I017
Day 6
tlast	(h)	24	24	24	24
tmax	(h)	0.5-2$	1-2$	1-2$	1-2$
Cmax	(ng/mL)	337	1320	4090	5950
AUClast	(h∙ng/mL)	816	4110	19800	46800
AUC∞	(h∙ng/mL)	914	4180	20200	47500
t½	(h)	6.41	3.69	3.92	3.70
Day 13		(4.18 mg/kg)
tlast	(h)	24-72$	48-72$	72	72
tmax	(h)	0.5-2$	0.5-2$	1-4$	2-4$
Cmax	(ng/mL)	213	1090	3770	6860
AUClast	(h∙ng/mL)	983	4990	24600	153000
t½	(h)	12.9	12.7	12.6	7.09
CPCA-carnitine
Day 6
tlast	(h)	4-8$	8-24$	24	24
tmax	(h)	1-2$	2	2-4$	4-8$
Cmax	(ng/mL)	192	447	1160	1810
AUClast	(h∙ng/mL)	710	2960	10500	25900
AUC∞	(h∙ng/mL)	n/a	n/a	9500	n/a
t½	(h)	n/a	n/a	6.53	n/a
Day 13		(4.18 mg/kg)
tlast	(h)	24-72$	72	72	72
tmax	(h)	2-4$	2-4$	4-8$	0.5-24$
Cmax	(ng/mL)	278	1020	1950	3120
AUClast	(h∙ng/mL)	3750	20700	53400	190000
t½	(h)	n/a	n/a	n/a	n/a

$: range;  _D : dose normalized to 1 mg/kg;  n/a: not applicable

Applicant's summary and conclusion

Executive summary:

Oral administration of the test substance via gavage to pregnant New-Zealand rabbits from Day 6 to Day 13 post-coitum did not lead to toxicologically relevant clinical signs up to a dose level of 32 mg/kg bw. Under these test conditions, reduced food intake was noted associated with feces reduction at a dose level of 60 mg/kg bw.
After oral administration the test substance was rapidly absorbed. The AUC values of the test substance increased with increasing dose level in an unproportional manner. From a dose level of 5 to 60 mg/kg bw of the test substance the AUC of it increased approximately 1900 and 7400 times on Days 6 and 13 post-coitum, respectively. The metabolites also displayed an overproportional increase from dose levels of 5 to 15, from 15 to 30 and from 30 to 60 mg/kg bw, although the relative increase of the internal dose over the oral dose was less pronounced for
these metabolites than for the parent compound. The test substance and the metabolites M440I001 and M440I017 displayed comparable concentration time curves after single and repeated administration. The metabolite CPCA- carnitine displayed a concentration plateau level with a minor decrease in concentration after repeated administration at the highest dose level.
Day 6 and Day 13 post-coitum, comparable exposures were noted for the parent compound and its metabolites at dose levels of 5 to 32 mg/kg, and no accumulation was observed, except for the metabolite CPCA-carnitine where slight accumulation was observed. At 60 mg/kg, higher exposures, in terms of AUC, for the test substance and its metabolites were noted on Day 13 postcoitum compared with Day 6 post-coitum indicating saturation kinetics at this dose level.
When the test substance is orally dosed to pregnant rabbits, the compound is rapidly absorbed. The internal dose of the test substance and its metabolites M440I001 and M440I017 (as indicated by their respective AUCs) showed an over-proportional increase in internal exposure from oral dose levels of 5 to 15, from 15 to 30 and from 30 to 60 mg/kg bw after single and repeated administrations in pregnant New Zealand White Rabbits. These data clearly demonstrate saturation of kinetics with increasing dose. Under the applied test conditions, this effect starts at a dose level of 15 mg/kg bw.