Prometryn

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Feb 1989 to 23 Jan 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

other: Agricultural Chemicals Laws and Regulations, Japan (II) Testing Guidelines of Toxicology Studies Society of Agricultural Chemical Industry

Version / remarks:

1985

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Prometryn

EC Number:

230-711-3

EC Name:

Prometryn

Cas Number:

7287-19-6

Molecular formula:

C10H19N5S

IUPAC Name:

6-(methylsulfanyl)-N2,N4-di(propan-2-yl)-1,3,5-triazine-2,4-diamine

Test material form:

solid: particulate/powder

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Tif: RAI f

Remarks:

SPF

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: about 9 weeks of age for females for the absorption and distribution kinetics study (Group E, F1 and F2) and about 10 weeks of age for females in the biliary excretion study (Group G)
- Weight: about 200 g for females for the absorption and distribution kinetics study (Group E, F1 and F2) and about 230 g for females in the biliary excretion study (Group G).
- Housing: The animals were individually kept in the following cages:
Group E: All-glass metabolism cages (Cage-System 1), suitable to anaesthetize the animals with O2/CO2 prior the collection of blood
Group F1, F2: Open plexiglass metabolism cages
Group G: Non-restrictive open plexiglass metabolism cages (Cage-System 3) suitable for the collection of bile.
Control Animals: Cage-System 2.
- Diet: The animals were allowed free access to certified standard diet, except the night before dosing in the morning. Analytical values for nutritive ingredients and contaminants in the food were provided by the manufacturer. Analytical results are filed at the testing facility.
- Water: Tap water was offered ad libitum at all times. The water quality was in compliance with the "Schweizerische Lebensmittel- gesetz" Routine chemical and bacterial analyses are conducted periodically by the water authority. In the postoperative phase the bile-duct cannulated rats received tap water containing 5% glucose 0.9% NaCl and 0.05% KCl.
- Acclimation period: Two to 5 days to the laboratory environment including 1 day to the metabolism cages under test conditions (Groups E, F1 and F2). The animals of Group G were acclimatized for 2 days to the laboratory environment and after bile duct cannulation kept for at least 1.5 hour in the non-restrictive metabolism cages (Cage-System 3) for recovery and adaptation before dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 46 - 66
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Hi-Sil suspended in 0.5% aqueous CMC

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with Hi-Sil and suspended in 0.5% aqueous CMC5. The ratio of Hi-Sil to the test substance was 1.5:1 (w/w). The volume of dosing suspension was about 1 mL per animal. One dose level was used. Each animal received 1 mL (Group E, F1, F2) or 0.9 mL (Group G) of the respective administration suspension by stomach tube. The control animals received a single oral dose of the administration vehicle without test substance.

Duration and frequency of treatment / exposure:

Single oral dose

Doses / concentrations

Dose / conc.:

0.5 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

3 to 10 animals per group. See Table 1 in "Any other information on materials and methods incl. tables" for more information.

Control animals:

yes, concurrent no treatment

Details on dosing and sampling:

Urine, faeces, and bile were individually collected for two day. Blood samples were taken during 14 days (see below). Blood was taken from the ophthalmic venous plexus (orbital sinus) of the anaesthetized animals (Group E). Anaesthesia was induced using a mixture of O2/CO2 (35 % / 65 %). The vessels used for collecting urine and bile were surrounded by solid carbon dioxide. Faeces was collected at ambient temperature. At the end of the collection period (Group G) the cages were rinsed thoroughly with water/ethanol 1/1 (v/v) = > Cage wash.
The animals were killed by exsanguiation after anaesthesia with carbon dioxide in a dessicator. Blood was collected into heparinized tubes. The following tissues and organs were taken from the animals of Group FI and F2 and weight prior to analysis: Bone, Brain, Fat (abdominal), Ovaries, Uterus, Heart, Kidneys, Liver, Lungs, Plasma, Red Blood Cells, Skeletal muscle, Spleen, Residual carcass.

The following samples were taken from the individual animals:
Group E:
- Blood: 1, 2, 4, 8, 12, 24, 32 hours, 2, 5, 9, and 14 days after administration (determination of t-max and tmax/2).
Group F1:
- Tissues: Eight hours (t-max) and 48 hours after administration the above listed tissues and organs were taken for analysis
Group F2:
- Tissues: Five days and 11 days (t-max/2) after administration the above listed tissues and organs were taken for analysis
Group G:
- Urine: 0 - 24, 24 - 48 h
- Faeces: 0 - 24, 24 - 48 h
- Bile: 0 - 0.5, 0.5 - 1, 1 - 2, 2 - 4, 4 - 8, 8 - 17, 17- 24, 24 - 41, and 41 - 48 h.

Urine, faeces, bile, tissues and organs were kept frozen, whole blood and red blood cells were kept refrigerated and cage wash was kept at ambient temperature until analysis.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

About 90% of the dose were absorbed from the gastrointestinal tract into the general circulation, as estimated from the amount biliary and renally excreted.

Main ADME results 2

Type:

distribution

Results:

Highest residue levels were found in red blood cells followed by organs with high blood perfusion: lung, spleen, heart and liver. Tissues residue levels were slightly lower in males compared to females.

Main ADME results 3

Type:

metabolism

Results:

The major metabolite fraction (0.9% of the dose) present in the faeces extract of the bile-duct cannulated rats corresponded to unchanged test substance.

Main ADME results 4

Type:

excretion

Results:

Excretion in bile duct-cannulated rats was 67 %, 24 % and 4 % via the bile, urine and faeces, respectively within two days, hence a significant part of amount excreted into faeces was absorbed and re-eliminated into intestinal tract by biliary excretion.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Within 48 hours after an oral administration at a dose level of 0.5 mg/kg about 90 % (mean value) of the dose were absorbed from the intestinal tract into the general circulation, as estimated from the amount biliary and renally excreted. Hence a significant part of the amount eliminated via the faeces by rats was absorbed and reentered the intestinal tract by biliary excretion. The blood kinetics of the animals in Group E confirmed the fast absorption rate of the orally administered test substance from the intestinal tract. The maximum blood level (tcmax ≈ 0.25 ppm) was reached about 8 hours after administration. However a concentration of 0.2 ppm was already reached 1 hour after dosing. The blood concentrations declined to one half of the maximum value (tcmax/2) about 11 days after administration.

Details on distribution in tissues:

See Table 2 in "Any other information on results incl. tables" for the distribution of test substance equivalents in vivo.

Details on excretion:

The bile flow from all animals of Group G was constant during the first 24 hours after administration (mean value of about 1.0 mL/h). Thereafter the bile flow decreased to a mean value of 0.6 mL/h during the last interval (41 to 48 hours after dosing). The orally administered radioactivity was fast excreted as almost 80 % were eliminated within the first 24 hours, by far the major portion with the bile. Within 48 hours of about 67 %, 24 %, and 4 % of the administered dose were excreted with the bile, urine, and faeces, respectively.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

URINE
After clean-up the pooled urine of the bile-duct cannulated rats (eluate Ul/f-2, corresponding to 22.3 % of dose) was analyzed by HPLC. The chromatography revealed 16 metabolite fractions. None of the metabolite fractions corresponded to unchanged test substance. One of the major metabolite fraction (U14, accounting for 3.9 % of the administered dose) behaved on HPLC and TLC like metabolite M6, i.e. the mercapturic acid derivative of the test substance. The most nonpolar metabolite fraction U15 (0.5 % of the dose) co-chromatographed with metabolite M4, the mono-dealkylated derivative of the test substance.

FAECES
The purified faeces extract of the bile-duct cannulated rats (Fl/f-lV3-2, corresponding to 2.2 % of the dose) was analyzed by one-dimensional TLC. The low concentration of radioactivity in the faeces extracts did not allow a proper co-chromatography with the reference compounds. Nevertheless the major metabolite fraction F8, representing more than 40 % of the extracted radioactivity (corresponding to 0.9 % of the dose), behaved in different TLC systems like unchanged test substance.

BILE
After clean-up the pooled bile (Gl/f-2) was analyzed by HPLC. The chromatography revealed 16 metabolite fractions. None of the metabolite fractions corresponded to unchanged test substance.
The major metabolite fraction Gil - representing 10.3 % of the administered dose - behaved in HPLC and TLC systems like metabolite M7 i.e. the glutathione conjugate of test substance. The most nonpolar metabolite fraction G15 (6.2 % of dose) co-chromatographed with metabolite M4, the mono-dealkylated derivative of test substance. The metabolite fraction G3 (4.6 % of dose) showed the same retention time (HPLC) as metabolite M13, the bis-dealkylated diamino derivative of test substance.

Any other information on results incl. tables

VERIFICATION OF FORMULATION STABILITY

TLC analysis revealed that the test substance was stable in the administration suspension. At the time of dosing the test substance represented > 95 % of the radioactivity, except for the bile fistula experiment (Group G: 94.7 %).

Table 1. Single oral doses administration

	Number and sexof animals	Administered sose
		[mg/kg]	kBq	(/tCi)
Group E	3 females	0.49-0.50	117	(3.2)
Group F1	10 females	0.50-0.56	118	(3.2)
GroupF2	10 females	0.49-0.54	115	(3.1)
GroupG	5 females	0.50-0.53	139	(3.8)

 

Table 2: Tissue Residues

Tissue residues in ppm test substance equivalents						Half life time [d]
Group	F1		F2
Necropsy after administration [d]	1/3	2	5	11		2-5	5-11
Dose [mg/kg]	0.53	0.54	0.51	0.51		t 1/2*	t 1/2 #
Spleen	0.1186	0.0937	0.041	0.0547		2.5	n a
Liver	0.2411	0.0975	0.0357	0.0235		2.1	9.9
Fat	0.0328	0.0098	0.0024	< LQ		1.5	n a
Kidneys	0.2133	0.0663	0.0263	0.0207		2.2	17.4
Muscle	0.0286	0.0103	0.0053	0.0034		3.1	9.4
Brain	0.0124	0.0076	0.0041	0.0035		3.4	26.3
Heart	0.0675	0.0427	0.0228	0.0171		3.3	14.5
Lungs	0.1245	0.1067	0.0581	0.048		3.4	21.8
Bone	0.0449	0.0145	0.0064	0.0046		2.5	12.6
Plasma	0.029	0.0053	0.0028	0.0017		3.3	8.3
Ovaries	0.0651	0.0293	0.012	0.0161		2.3	n.a.
Uterus	0.0485	0.0118	0.0058	0.0044		2.9	15.1
Red Blood Cells	0.7259	0.7845	0.4401	0.3661		3.6	22.6
Carcass	0.3163	0.0161	0.0053	0.0053		3.2	9.0

* Phase 1: calculated from the residue data at 2 and 5 days after administration and assuming a first order kinetics

# Phase 2: calculated from the residue data at 5 and 11 days after administration and assuming a first order kinetics

n.a. = not applicable

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test substance was fast absorbed and rapidly excreted after single oral administration to female rats at a dose level of about 0.5 mg/kg body weight. About 42 % of the absorbed dose was eliminated into faeces and 50 % was eliminated into urine, the pattern of elimination was not dependent on sex or dosing regimen. Highest residue levels were found in red blood cells followed by organs with high blood perfusion: lung, spleen, heart and liver. Tissues residue levels were slightly lower in males compared to females. The major metabolite fraction (0.9 % of the dose) present in the faeces extract of the bile-duct cannulated rats corresponded to unchanged test subbtance. Excretion in bile duct-cannulated rats was 67 %, 24 % and 4 % via the bile, urine and faeces, respectively within two days, hence a significant part of amount excreted into faeces was absorbed and re-eliminated into intestinal tract by biliary excretion.

Executive summary:

The fate of the test substance, the active ingredient of the herbicide proposed for the control of a wide range of annual broadleaved weeds in various crops, was investigated in female rats using [U-14C]triazine labeled test substance. About 90 % of an oral administered dose (0.5 mg/kg) were absorbed from the gastrointestinal tract into the general circulation. Within 48 hours about 67 %, 24 %, and  4% of the administered dose were excreted with the bile, urine, and faeces, respectively, by bile-duct cannulated rats. The residual radioactivity was determined 8 hours (time point of maximum concentration of radioactivity in the whole blood), 2 days, 5 days, and 11 days (one half of maximum blood concentration) after dosing in spleen, liver, fat, kidneys, muscle, brain, heart, lungs, bone, plasma, ovaries, uterus, red blood cells, and in the remaining carcass. Five days after an oral administration of 0.5 mg/kg body weight the tissue residues were below 0.06 ppm test substance equivalents, except in red blood cells (0.44 ppm). Within eleven days after dosing the tissue residues declined to values < 0.02 ppm, except in red blood cells (0.37 ppm), spleen, lungs (0.05 ppm), liver and kidneys (0.02 ppm). The residues in red blood cells corresponded to 3.9 %, 4.3 %, 2.6 %, and 2.3 % of the dose at 8 hours, 2 , 5, and 11 days after administration, respectively. The half life time for the depuration of the residues from the red blood cells was calculated to be 4 days for phase 1 (2 to 5 days after administration) and 23 days for the slower phase 2 (5  to 11 days after dosing). These two phases were also observed in the other tissues and organs, demonstrating a non-linear behavior of the depletion kinetics (assuming first order kinetics). The relatively high residues in the red blood cells and the pronounced slow depuration of these residues is characteristic for methylthio triazines in rats. This class of substances form stable derivatives with rodent and chicken haemoglobins, but do not react with human haemoglobins.

One of the major urinary metabolite fractions co-chromatographed with an authentic reference compound. This mercapturic acid derivative accounted for about 4 % of the administered dose. Another urinary metabolite fraction (0.5 % of dose) behaved like metabolite M4. The major metabolite fraction (0.9 % of the dose) present in the faeces extract of the bile-duct cannulated rats corresponded to unchanged test substance. The major biliary metabolite fraction - accounting for 10 % of the administered dose - cochromatographed with an authentic reference compound. Two other major metabolite fractions cochromatographed with metabolite M4 (6.2 % of dose) and metabolite M1, the mono- and bis- dealkylated derivatives of the test substance, respectively. Besides mono- and bis-dealkylation at the amino groups the glutathione pathway play an important role for the elimination of the test substance.