1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According guideline; performed under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Objective of study:

absorption

excretion

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

EC Number:

603-837-5

Cas Number:

134605-64-4

Molecular formula:

C20H18ClF3N2O6

IUPAC Name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Test material form:

not specified

Details on test material:

- Radiochemical purity: 97.7%
- Specific activity: 41.6 microCi/mg
- Locations of the label (if radiolabelling): The location of the site of 14C label were reported within the study.
- Expiration date of radiochemical substance (if radiolabelling): Not reported.
- Stability under test conditions: Stable under the conditions of the study
- Storage condition of test material: Freezer temperatures - 20°C

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data presented in this report. Hazleton report HWI 6117-331 cited for these data (Single oral dose biliary excretion study with phenyl-14C-test substance in bile duct-cannulated rats, Cheng T).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: PEG/EtOH/water (5/3/2)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Very low water solubility
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): Not reported

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Not reported. Stability of dosing solutions verified by HPLC at Corning Hazleton, Inc. during the biological phase of the study.

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

Remarks:

Doses / Concentrations:
0.5 mg /kg body weight

No. of animals per sex per dose / concentration:

4 per sex

Control animals:

no

Details on study design:

Due to the very low water solubility of the test substance, it was not possible to use an intravenous dose group to determine the absorption of test substance in male and female rats. Consequently, this bile duct-cannulation study was conducted to support a previous rat study (The metabolism of phenyl-14C-test substance in rats. Characterization and identification of metabolites, Emrani J, 1997) and to determine the absorption of 14C-test substance that had been administered orally to male and female rats.

Secondary objectives of this study were to compare the extent of biliary elimination between male and female rats and to characterize the metabolites in bile.

Four male and four female rats which had been duodenally cannulated were treated orally with a single dose of phenyl-14C-test substance. The test substance had a specific activity of 41.6 uCi/mg and was dissolved in PEG/EtOH/water (5/3/2). The actual average dose rate was 0.5 mg test substance per kg body weight. Bile, urine and feces were collected from all animals at specified time intervals until the rats were sacrificed at 48 hours after dose.

Details on dosing and sampling:

As above.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on excretion:

A detailed analysis of the in-life results and 14C-balance data for cannulated rats dosed with phenyl-14C-test substance is presented in the Hazleton report, Single Oral Dose Biliary Excretion Study with Phenyl-14C-test substance in Bile Duct-Cannulated Rats (Cheng T). The average percent of 14C-dose recovered in male and female bile at varying sampling intervals is summarized in Table 1, as are the average total recoveries. The average percent of 14C-dose recovered in male and female urine and feces at varying sampling intervals is summarized in Table 2.

The total mean recovery of radioactivity at 48 hours was 101% for both males and females. For both genders, nearly all of the excreted radioactivity was eliminated in the 0-24 hour interval. The radioactivity in the bile decreased sharply after the 4-8 hour interval.

There was a slight sex related difference in the elimination patterns, with biliary excretion favored in males (78.7%) slightly more than in females (74.3%) and urinary excretion being exactly the opposite (1.55% for male, and 7.23% for female). However, in both males and females the biliary excretion was the predominant route of elimination. The total average elimination of radioactivity in urine and bile was 80.25% for males and 81.53% for females. Accordingly, the test substance was about 80% absorbed at the 0.5 mg/ml oral dose level, regardless of rat gender.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Pooled composites of bile from the first four-hour collection interval from each rat were prepared to simplify comparisons between male and female bile. The composites of male and female bile samples were profiled and quantified by 2D-TLC. Qualitatively, the profiles were similar for both genders.

No parent compound was observed in the bile. The chromatographic profile of the bile was consistent with the metabolite profile of the excreta from a previous metabolism study in rats (The metabolism of phenyl-14C-test substance in rats, Characterization and identification of metabolites. Emrani J). The major biliary metabolites were shown to have the same 2D-TLC behavior as available standards. Bile samples were co-chromatographed with selected standards and also with high dose male feces extract to verify the applicability of the TLC assignments. The pooled bile profiles were calculated on a percent of dose basis to compare biliary metabolites in males and females. The excess percent of dose in male bile was largely due to the CGA-293731 metabolite. The metabolites CGA-293730 and CGA-368221 were present in slightly higher abundance in the female bile than in male bile.

The major metabolite of test substance observed in bile was CGA-293731(60.9% - 73.3% dose ). Minor metabolites included HO-CGA-293731 (1.5% - 3.0%) and CGA-368221(2.2% - 4.5%). Several minor (polar) components present at low levels (≤2% dose) in the male and female bile samples were characterized as glucuronide conjugates of the major metabolites.

This study showed that more than 80% of the total dose was absorbed in both male and female rats at the low (nominal 0.5 mg/kg} dose level. Characterization showed that the major metabolites in the bile were the same as the metabolites identified in the excreta from the aforementioned rat study (Emrani J). Minor components in the bile were characterized as conjugates of the known major metabolites. See Table 3.

Any other information on results incl. tables

Table 1: Average percent of radioactivity in bile at each sampling interval for male and female rats

Collection Interval (hours)	Male Mean	SD	Female Mean	SD
0 - 4	59.70	5.10	63.50	3.29
4 - 8	9.19	1.74	5.70	0.73
8 - 12	2.99	0.29	2.53	1.26
12 - 24	4.20	1.13	1.98	1.16
24 - 48	2.62	2.00	0.67	0.85
Total	78.70	1.61	74.38	1.89
Total Recovered	101.00	0.43	101.00	0.59

Data extracted from report HWI 6117 -331

SD: Standard Deviation

Total recovered includes bile, urine and feces, cage wash, cage wipe and carcass

Table 2: Average percent of radioactivity in urine and feces at each sampling interval for male and female rats

Sample/Collection Interval (hours)	Male Mean	SD	Female Mean	SD
Urine
0 - 24	1.42	0.81	6.94	1.68
24 - 48	0.13	0.05	0.28	0.18
Urine Total	1.55	0.84	7.23	1.64
Feces
0 - 24	17.10	1.24	17.40	1.58
24 - 48	2.83	1.07	1.53	1.09
Feces Total	19.90	1.22	18.90	2.19
Total Excreted	21.45	1.19	26.13	2.36

Data extracted from report HWI 6117 -331

SD: Standard Deviation

Total excreted in urine and feces

Table 3: Quantitative 2D-TLC metabolite distributions for the 0 - 4 hour pooled bile composites from male and female rats (expressed as percent of assigned ¹⁴C and as percent of dose)

	% of assigned ^14C zone		% of dose basis  *
	Male	Female	Male	Female
POD in bile 0 - 4 hrs			63.5	59.7
Total POD in bile			74.3	78.7
U1  test substance	ND	ND	ND	ND
U2  CGA-293731	82.0	93.1	60.9	73.3
U3 CGA-380962	ND	ND	ND	ND
U4  HO-CGA-293731	4.1	1.9	3.0	1.5
U5  CGA-368221	6.1	2.8	4.5	2.2
U6  CGA-293730	0.5	ND	0.4	ND
U7  Acetic Acid Conj-CGA-293731	ND	ND	ND	ND
U8  CGA-321432	ND	ND	ND	ND
U9 Conjugate	1.9	0.5	1.4	0.4
U10 Conjugate	1.9	0.6	1.4	0.5
U11 Conjugate	0.4	0.5	0.3	0.4
U12 Conjugate	0.4	0.6	0.3	0.5
U13 Conjugate	0.6	ND	0.4	ND
U14 Conjugate	2.2	ND	1.6	ND
Recovery	100.1	100.0
POD Assigned			74.4	78.7

POD: Percent of dose

Zones U9 - U14 are primarily conjugates of the known metabolites

* Assumes 0 - 4 hrs is representative of total bile residues

% of dose basis = % of assigned zone x total POD in bile

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Male and female rats were successfully cannulated and dosed with 14C-test substance. Bile, urine and feces samples were collected over 48 hours and the balance data allowed the absorption of 14C-test substance to be assessed (as percent of dose in urine and bile) at the low dose level of 0.5 mg/kg. Results showed that over half of the 14C was absorbed with the absorption (as percent of dose in bile and urine) being comparable for males (80.25%) and females (81.53%). Profiling of selected male and female bile samples and composites showed that the major metabolites (95%) in the bile are the same as the major metabolites in the excreta. The 4-6 minor metabolites (a total of 5%) in the bile appear to be conjugates of the major metabolites. No parent compound was present in the bile of either the male or female rat. The profiles indicated that male and female bile contained common metabolites that differed in both relative abundance and percent of dose.

Executive summary:

The study was performed in accordance with EPA OPP 85-1. Four male and four female rats which had been duodenally cannulated were treated orally with a single dose of phenyl-14C-test substance. The test substance had a specific activity of 41.6 μCi/mg and was dissolved in PEG/EtOH/water (5/3/2). The actual average dose rate was 0.5 mg test substance per kg body weight. Bile, urine and feces were collected from all animals at specified time intervals until the rats were sacrificed at 48 hours after dose.

After 48 hours, the average recovery in bile and excreta was 100.15% for males and 100.43% for females. Elimination was rapid with the majority of the dose being excreted within 24 hours. The average percent of dose eliminated by various routes was as follows (male/female): Bile: 78.70 / 74.30; Urine: 1.55 / 7.23; Feces: 19.90 / 18.90; Total: 100.15 / 100.43. The average total amount of absorbed dose (urinary plus biliary elimination) was comparable for males (80.25%) and females (81.53%). There was a slight gender related difference in the primary excretion pattern as seen above.

Pooled composites of bile from the first four-hour collection interval from each rat were prepared to simplify comparisons between male and female bile. The composites of male and female bile samples were profiled and quantified by 2D-TLC. Qualitatively, the profiles were similar for both genders.

No parent compound was observed in the bile. The chromatographic profile of the bile was consistent with the metabolite profile of the excreta from a previous metabolism study in rats (The metabolism of phenyl-14C-test substance in rats, Characterization and identification of metabolites. Emrani J). The major biliary metabolites were shown to have the same 2D-TLC behavior as available standards. Bile samples were co-chromatographed with selected standards and also with high dose male feces extract to verify the applicability of the TLC assignments. The pooled bile profiles were calculated on a percent of dose basis to compare biliary metabolites in males and females. The excess percent of dose in male bile was largely due to the CGA-293731 metabolite. The metabolites CGA-293730 and CGA-368221 were present in slightly higher abundance in the female bile than in male bile.

The major metabolite of test substance observed in bile was CGA-293731(60.9% - 73.3% dose ) . Minor metabolites included HO-CGA-293731 (1.5% - 3.0%) and CGA-368221(2.2% - 4.5%). Several minor (polar) components present at low levels (≤2% dose) in the male and female bile samples were characterized as glucuronide conjugates of the major metabolites.

This study showed that more than 80% of the total dose was absorbed in both male and female rats at the low (nominal 0.5 mg/kg) dose level. Characterization showed that the major metabolites in the bile were the same as the metabolites identified in the excreta from the aforementioned rat study (Emrani J). Minor components in the bile were characterized as conjugates of the known major metabolites.