2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1970

Report date:

1970

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The labelled compound was dosed to mice, rats, guinea-pigs, dogs, and rhesus monkeys. The absorption, distribution, metabolism and excretion of the test substance was studied.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

EC Number:

248-383-5

EC Name:

2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Cas Number:

27277-00-5

Molecular formula:

C9H13N5O

IUPAC Name:

2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Test material form:

solid: particulate/powder

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

other: mice, rats, guinea-pigs, dogs, and rhesus monkeys.

Strain:

not specified

Administration / exposure

Route of administration:

other: oral gavage or capsule /

Details on exposure:

The labelled test substance was dosed orally to small animals by catheter as an aqueous solution and to dogs and monkeys in capsules.

Duration and frequency of treatment / exposure:

Animals were dosed once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: mg/kg bw

Remarks:

Tissue distribution: Male guinea-pigs

Doses / concentrations 2

Dose / conc.:

0.5 other: mg

Remarks:

Tissue distribution: Mice, oral and I.V. administration / equivalent to approximately 25 mg/kg bw

Doses / concentrations 3

Dose / conc.:

1 other: mg/kg

Remarks:

Comparative serum levels: mice, rats, guinea pigs

Doses / concentrations 4

Dose / conc.:

2.1 other: mg/kg bw

Remarks:

ADME: Rat

Doses / concentrations 5

Dose / conc.:

1.7 other: mg/kg bw

Remarks:

ADME: Guinea-Pig

Doses / concentrations 6

Dose / conc.:

12.9 other: mg/kg bw

Remarks:

ADME: Mouse

Doses / concentrations 7

Dose / conc.:

8.4 other: mg/kg bw

Remarks:

ADME: Mouse

Doses / concentrations 8

Dose / conc.:

0.25 other: mg/kg bw

Remarks:

ADME: Dog

Doses / concentrations 9

Dose / conc.:

0.04 other: mg/kg bw

Remarks:

ADME: Rhesus Monkey

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (distribution): Urine, faeces, bile, blood and several organs (guinea pig: Liver, lung, heart, spleen, kidney, muscle, fat, and brain).
- Time and frequency of sampling (distribution), guinea-pigs: 1/4, 1/2, 3/4, 1, 1.5, 2, 3, 4, 6, 24, 48 hours after dosing
- Time and frequency of sampling (distribution), mice: 1/4 and 1 hour after oral dosing and 1/12, 1/4, 1/2, 1, 3, 5, and 24 hours after intravenous dosing
- Time and frequency of sampling (ADME), all species: 4, 5, 6, 7, 24, 31, 48, 55, 72, 96 hours (urine and faeces)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, bile
- From how many animals: all animals/species
- Time and frequency of sampling (ADME), all species: 4, 5, 6, 7, 24, 31, 48, 55, 72, 96 hours (urine and faeces)
- Method type for identification: thin-layer chromatography (T.L.C.) in chloroform/methanol (9:1) on merck silica GF plates, NMR, mass spectrometry, DAD

COMPARATIVE SERUM LEVELS OF THE LABELLED TEST SUBSTANCE AND ITS METABOLITES
- Tissues and body fluids sampled: blood
- From how many animals: Eight male mice, five male rats, and five male guinea pigs were each dosed orally (1.0 mg/kg) with the test substance. The sera from the mice were pooled and the sera from the other ten animals examined separately.
- Time and frequency of sampling: Animals were all killed 1 hour after dosing and blood removed.
- Other: The serum samples were counted to determine the total radioactivity levels and aliquots extracted with chloroform. The extracts were counted and also examined by T.L.C. in order that the amounts of each labelled component present could be determined. The serum levels of the test substance and the hydroxylated metabolite were calculated. Duplicate serum samples from the guinea-pigs and rats, and the pooled sera from the mice were assayed by the method based on the fluorescence of the test substance.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

ADME IN ALL SPECIES (SINGLE EXPOSURE)
The test substance is well absorbed after oral administration in all test species.

Details on distribution in tissues:

TISSUE DISTRIBUTION OF RADIOACTIVE MATERIAL, GUINEA-PIGS
- The specific activity of the labelled test substance was 15,060 dpm/µg. - Serum and tissue levels were comparatively steady over the period 1/4 to 4 hours after oral administration, the maximum levels occurring at about 1 hour.
- The half-life of radioactivity in serum is of the order of 4 hours. Higher levels of radioactivity occur in bile, liver, and kidney, but it should be noted that even at 6 hours the comparatively high level in bile corresponds to only ca. 8 µg/mL. The brain level has dropped by 6 hours and was not detectable at 24 hours. Only very low levels of radioactivity were present at 28 hours in all tissues examined. See Table 3 in ‘any other information on results incl. tables’ for details.

WHOLE-BODY AUTORADIOGRAPHY, MICE
- The specific activity of the labelled test substance was 6.8 µCi/mg. - Radioactivity was distributed throughout all the tissues. It was almost completely cleared from the animal at 24 hours.
- At 15 min the orally dosed animal had very high activity in the stomach contents with some labelling of urine and bile, but no activity was detected in either liver or kidney at this time. At 1 hour after an oral dose, much activity still remained in the stomach but some absorption had occurred. Activity was detected in all tissues with high concentrations in renal medulla, urine and bile.
- At 5 min. after an i.v. dose, all tissues, including brain, intestinal wall and urine, were labelled. A small amount of activity was present in the contents of the pyloric region of the stomach. Levels of activity in all tissues, except bile, duodenal contents and urine, decreased progressively over the first hour. At one hour the nasal secretions were also labelled. At 3 and 5 hours all tissues were still labelled with high concentrations in bile, duodenal contents, bladder contents and nasal secretions. Very little activity was present in fully formed faecal pellets. After 24 hours, very little activity remained in the animal but the bladder wall was labelled.
- Since only a small proportion of dosed radioactivity is recovered in mouse faeces after oral administration, and since after the i.v. dose there is evidence of extensive biliary excretion, re-absorption from the gut is indicated in this species.

Transfer into organs

Transfer type:

blood/brain barrier

Remarks:

Guinea-pigs / Mice

Observation:

not determined

Details on excretion:

EXCRETION
- The radioactivity was measured in urine and faeces and exhaled air of the dosed animals.
- 96 hours after dosing total excretion of radioactivity was 104.6, 90.0, 90.2, 90.9, 104, 100.6% in the rat, guinea-pig, mouse, mouse, dog, and rhesus monkey, respectively.

FAECAL AND BILIARY EXCRETION
- The rat is the only species studied in which a significant proportion (43%) of an oral dose is excreted in faeces. Homogenisation of rat faeces in methanol afforded an extract containing 13% of the faecal radioactivity. The extract was shown by T.L.C. to contain a component having the same T.L.C. properties as the test substance (23%) and unresolved polar compounds.
- The labelled test substance was dosed orally (1.3 mg/kg bw) to a male rat whose bile duct had been cannulated. In the first 6 hrs, 20% of the dose was excreted in bile and by 44 hours, 58% of the dose. At the same dose level a male guinea-pig passed only 2.5% in bile in a 5.5 hour experiment the bile was found to contain the labelled test substance (15%), the hydroxylated metabolite (6%) and polar material (79%).

See Table 1 in ‘any other information on results incl. tables’ for details.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

URINARY METABOLITES
- Urine samples from different species were found to contain the amounts of unchanged test substance and its hydroxylated metabolite. See Table 2 in ‘any other information on results incl. tables’ for details.

SPECIES DIFFERENCES
- Urine samples from all species showed only two major fractions when examined by T.L.C.: the hydroxylated metabolite and polar (origin spot) material. The latter probably contains conjugates, but these have not been identified.
- The rat produces a minor urinary metabolite (5% of the material in urine) whose molecular weight corresponds to that of the test substance with an additional oxygen atom and an additional acetyl group. Its fluorescence spectrum (Ex nλ Max 285 nM; Em nλ max 389 nm) is identical with that of N-acetyl test substance. This minor metabolite has not been observed in urine from other species. But trace amounts of a labelled compound having the same T.L.C. properties have been found in chloroform extracts of mouse and guinea-pig serum.
- The mouse excretes a significant proportion (9.2, 8.4, and 4.7% in 3 experiments) of an oral dose (8 to 14 mg/kg bw) in exhaled air and in this respect differs from rat (1.6%) and guinea pig (none detected). This could indicate dealkylation, but the dealkylated compound has not been detected in urine from animals dosed orally with the test substance.
- As rat, mouse, guinea-pig, dog and rhesus monkey all produce the hydroxylated metabolite as the single major non-conjugated urinary metabolite, these species have at least one major metabolic pathway in common.

PROPERTIES OF URINARY HYDROXYLATED METABOLITE
- The hydroxylated metabolite has been isolated from a chloroform extract of mouse urine. It has not been synthesised. The structure of the metabolite was deduced from its mass spectrum and N.M.R. spectrum. The parent ion in the mass spectrum has m/e = 223, showing that the molecular formula differs from that of test substance by one oxygen atom. The N.M.R. spectrum shows that all the protons of the propyl side chain and the ring proton are intact. However, the signal due to the methyl group in the test substance (8.04 T) is replaced by a two-proton signal at 5.76 T, chemical shift being consistent with substitution at the carbon by an oxygen function.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

COMPARATIVE SERUM LEVELS OF THE LABELLED TEST SUBSTANCE AND ITS METABOLITES
- See any Table 4. in 'any other information on results incl. tables' for details.
- the total (radioactive) serum level in guinea-pig is about five to six times higher than those in rat and mouse.
- 96 % of the material in guinea-pig serum is extractable into chloroform at physiological pH, whereas 70 % is extractable from rat serum and only 55 % from mouse serum.
- A major component in the serum in all three species is the labelled test substance (guinea-pig 71 %, rat 57 %, mouse 34 %).
- The hydroxylated metabolite is a minor component in the serum of all three species (guinea-pig 5 %, rat 4 %, mouse 7 %).
- There is an excellent agreement in the two sets of results from the TLC method and the method based on the fluorescence of the test substance.See any Table 5. in 'any other information on results incl. tables' for details.

Any other information on results incl. tables

Table 1. Excretion of radioactivity following oral administration of the labelled test substance

Species	Rat (male)^a		Guinea-Pig (male)^b		Mouse (male)^c		Mouse (male)^d		Dog (male)	Rhesus monkey (female)
Dose mg/kg bw	2.1		1.7		12.9		8.4		0.25	0.04
Time (hr.)	U	F	U	F	U	F	U	F	U (only)	U (only)
4										37.7
5										51.3
6										58.5
7	23.3	2.6	11.8	0.6	15.3		21.8			65.7
24	55.0	21.5	63.5	1.8	66.3	9.1	67.7		100.6
31	57.3		69.0				70.6			98.6
48	59.4	41.6	73.6	11.8	69.6	10.6	74.4		103.9	100.4
55			74.1				75.0		104.0	100.6
72	59.7	42.9	74.8	14.3	70.0	11.0	76.9	5.6	104.0
96	59.8	43.2	75.0	15.0
Total (U + F)^e	104.6		90.0		90.2		90.9		104.0	100.6

a. 1.6% dose in exhaled air in the first 24 hr.

b. No radioactivity detected in exhaled air.

c. 9.2% dose in exhaled air in the first 24 hr.

d. 8.4% dose in exhaled air in the first 24 hr.

e. Included % dose in exhaled air where appropriate.

U = Urine

F = Faeces

 

Table 2. Proportions of the test substance and its metabolite in urine

Species	Dose mg/kg bw	Sample (hr)	% Dose in sample	% test substance^a	% hydoxylatedmetabolite^a
Rat (male)	2.1	7 - 24	32	9.6	14.8
Guinea-pig (male)	1.7	7 - 24	52	2.9	36.8
Dog (male)	0.25	0 - 24	100	1.0	33.0
Rhesus Monkey (female)	0.04	0 - 4	38	3.0	38.1
Mouse (male)	13.5	0 - 7	44	2.3	35.2

a: of the total radioactivity in the urine sample indicated.

Table 3. Distribution of labelled material in guinea-pigs following administration of the labelled test substance 1.0 mg/kg

Time (hr)	Serum	Blood	Bile	Liver	Lung	Heart	Spleen	Kidney	Muscle	Fat	Brain
¼	16.4^a	11.5	19.5	15.7	3.8	6.9	2.6	7.2	3.8	1.8	3.8
	100^b	70	119	96	23	42	16	44	23	11	23
½	16.0	11.7	24.2	13.0	5.7	3.9	3.0	6.6	4.3	2.2	3.6
	100	73	151	81	36	24	19	41	27	14	22
¾	16.6	12.9	33.4	14.9	7.9	7.6	3.2	7.6	5.5	2.6	4.8
	100	78	202	90	48	46	19	46	33	16	29
1	22.4	17.9	46.8	18.9	5.6	12.3	3.1	10.6	7.8	3.8	7.2
	100	80	209	84	25	55	14	47	35	17	32
1 ½	21.1	14.4	47.8	16.4	6.0	11.2	3.1	11.1	8.2	3.2	4.0
	100	68	226	77	29	53	14	53	39	15	19
2	21.4	14.7	57.7	16.5	6.4	8.5	2.9	11.2	11.2	2.9	4.8
	100	69	269	77	30	40	13	52	52	13	22
3	17.2	clotted	69.8	15.4	5.1	4.9	3.1	12.3	10.3	3.4	4.9
	100		406	90	30	29	18	72	60	20	28
4	17.5	11.6	78.2	14.2	6.5	7.7	2.9	10.7	5.4	2.9	4.2
	100	66	446	81	37	44	16	61	31	16	24
6	11.6	8.6	122.5	10.0	4.5	3.5	2.8	11.0	5.0	2.2	2.9
	100	74	1058	86	39	30	24	95	43	19	25
24	0.41	0.30	3.6	0.86	0.31	0.10	0.09	0.35	0.15	0.20	ND
	100	71	863	207	75	24	22	85	35	47
48	0.23	0.15	0.60	0.28	0.20	0.08	ND	0.23	ND	ND	ND
	100	60	256	119	85	32		98

a. Throughout dpm/mL or g x 10^-3

b. Throughout % serum level

ND: not detected

Table 4. Comparative serum levels in guinea-pig, rat and mouse

Species (all male)	Serum level dpm/mL^a	% extracted into chloroform^b	% composition of chloroform extract determined by TLC (A) and calculated % in serum (B)
			Test substance		Hydroxylated Metabolite
			A	B	A	B
Mouse^c	2852	55	62	34	13	7
Rat (average n=5)^d	3714 ± 75	70 ± 4		57 ± 3		4
Guinea-pig (average n=5)^d	18436 ± 648	96 ± 2		71 ± 2		5

a. Specific activity 15060 dpm/µg (as test substance)

b. At physiological pH

c. Values for serum pooled from 8 animals

d. ± S.E.

ND: Not detected (less than 1%)

Table 5. Comparison of serum levels of the test substance and hydroxylated metabolite determined by fluorescence and thin-layer chromatography

Species (all male)	Serum concentration of the test substance and its hydroxylated metabolite
		By fluorescence µg/mL	By T.L.C µg/mL
Guinea-pig	1	1.12	0.91
	2	1.07	0.94
	3	1.06	1.04
	4	1.04	0.99
	5	0.88	0.76
Rat	1	0.10	0.13
	2	0.12	0.16
	3	0.15	0.18
	4	0.12	0.14
	5	0.12	0.13
Mice (8 animals)		0.12	0.08

Applicant's summary and conclusion

Conclusions:

Interpretation of results: low bioaccumulation potential based on study results
The test substance is well absorbed after oral administration in mice, rats, guinea-pigs, dogs, and rhesus monkeys and excreted via urine and faeces as the parent compound or as its non-conjugated hydroxylated metabolite. The biological half life in dogs is < 3 hours.

Executive summary:

The test substance is well absorbed after oral administration in all species except rats. At least 70% of the dosed radioactivity is passed in urine within 48 hours. Rat differs from the other species in passing a large proportion (43%) of an oral dose in faeces. It has been shown that biliary excretion is of major importance in this species and whole body autoradiography indicates that biliary excretion and reabsorption occurs in mice. The test substance was extensively metabolised in all species studied. One single non-conjugated major metabolite occurs in the urine of all species, a metabolite in which the methyl group of the test substance has been hydroxylated. The sera from rat, guinea-pig, and mouse contain only small (4 - 7% of the total radioactivity in serum) of this metabolite; the one major component present being the test substance. The tissue distribution curves in guinea-pigs show that maximum levels of radioactivity in serum and brain occur at about 1 hour after oral administration. The serum and tissue levels were found to be steady over the period 1/4 to 4 hours after dosing. 48 hours after oral administration of the labelled test substance to a guinea-pig, the serum level was found to be 1 % of the maximum level at 1 hour and only very low levels of radioactivity were detected in liver, kidney, bile, lung and heart. The maximum serum level of the test substance is higher in guinea-pigs (0.87 µg/mL) than in rat (0.17 µg/mL) and mouse (0.06 µg/mL) after an oral dose of 1 mg/kg bw.