3-hydroxy-2-methyl-4-pyrone

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

The Metabolism of Ethyl Maltol and Maltol in the Dog

Author:

Hans H. Rennhard

Year:

1971

Bibliographic source:

J. Agr. Food Chem., Vol. 19, No. 1, 1971

Materials and methods

Objective of study:

excretion

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In a crossover design study, groups of two beagle dogs of each sex were given a single intravenous injection of 10 mg/kg bw maltol or ethyl maltol and urine samples were collected for 3, 6, 24, 48 and 72 hr. The second administration (crossover) was done 7 days after the first. The cumulative urinary excretion of ethyl maltol and maltol conjugates (sulfate and glucuronide) in percent of the administered dose was presented.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-2-methyl-4-pyrone

EC Number:

204-271-8

EC Name:

3-hydroxy-2-methyl-4-pyrone

Cas Number:

118-71-8

Molecular formula:

C6H6O3

IUPAC Name:

3-hydroxy-2-methyl-4H-pyran-4-one

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and of test material: Synthetic metabolites (sulphate, glucuronide) of Maltol were synthesised by the author according to methods in the literature and were considered pure compounds.

Radiolabelling:

no

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were maintained in metabolism cages and fed once daily; water was given ad libitum. This study was started with fresh (not prestressed) dogs.

Administration / exposure

Route of administration:

intravenous

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

IV study: Single dose once and then again 7 days later (crossover design)

Doses / concentrations

Dose / conc.:

10 other: mg/kg bw

Remarks:

IV study

No. of animals per sex per dose / concentration:

IV study: 2 males and 2 females

Control animals:

no

Positive control reference chemical:

None

Details on dosing and sampling:

TOXICOKINETIC STUDY
Urine samples were collected for 3, 6, 24, 48 and 72 hr after each administration (7 days apart).

METABOLITE CHARACTERISATION STUDIES
The protocol also decribes ethyl maltol experiments.

Identification and Synthesis of Metabolites.
Urine excreted over a 24 hr period following intravenous administration of ethyl maltol (10 mg per kg) was examined by paper chromatography (system D, detector reagent a). No unchanged ethyl maltol (Rf 0.7) was found. However, treatment of the urine with mineral acid (refluxing equal volumes of urine and 6N sulfuric acid for 2 hr in oil bath) resulted in the formation of substantial quantities of ethyl maltol. This observation led to the conclusion that ethyl maltol is excreted as one or more conjugates, possibly as ethereal sulfate and/or β-glucuronide. Treatment of the urine with β-glucuronidase (0.2M pH 5 sodium acetate-acetic acid buffer, 1 mg β -glucuronidase per ml of urine, incubated 29 hr at 46C) also regenerated ethyl maltol, indicating the presence of a p-glucuronide. Sequential treatment first with 0.25N hydrochloric acid (15 min at 100C), known to hydrolyze sulfates but not β -glucuronides , then with β - glucuronidase or 6N sulfuric acid (conditions described above) resulted in incremental regeneration of ethyl maltol in both steps, and thus implicated both sulfate and glucuronide as conjugates. Final confirmation of the metabolic route was obtained by identification of the urine components of treated animals with synthetic metabolites.

Hydrolysis of the Metabolites and Colourimetric Determination of the 2-Alkyl 3-Hydroxy-1,4-pyrones Formed.
In order to be amenable for colourimetric determination as their ferric complexes, ethyl maltol and maltol were regenerated from the metabolites by graded acid hydrolysis. Heating the ethereal sulfates in 0.25N sulfuric acid for 15 min at 100" C caused their complete hydrolysis, whereas the glucuronides proved to be stable under these conditions. Hydrolysis of the glucuronides required refluxing in 3N sulfuric acid for 2 hr. Ethyl maltol and maltol were determined colourimetrically as their ferric complexes at 520 nm. Reagent: 1%ferric ammonium sulfate in 0.36M sulfuric acid. Concentrations were read from a standard curve linear in the range of 0.5 to 5 mg %

Isolation of Ethyl Maltol and Maltol from Urine.
The ethyl maltol and maltol formed by hydrolysis of the metabolites was extracted five times from the acid solution with chloroform. After replacement of the chloroform by 40% aqueous methanol the pyrones were determined as described above. Since chloroform extracts of normal urine heated with an equal volume of 6N sulfuric acid undergo a slight colour change in the presence of ferric ions, the intensity of this background colour was determined experimentally and applied as a correction. Mean value of 23 determinations. Absorbance at 520 nm 0.015 (range: 0.005 to 0.028). The colour inherent to excess reagent was found to be negligible (transmission vs water: 99%. Synthetic metabolites were added to normal dog urine, which was then put through the described two step hydrolysis, and extracted with chloroform after each step.

Recoveries: Metabolite Ethyl Maltol Maltol
Sulfate 83 83.5
Glucuronide 90.5 85

These recovery factors, as well as the background colour factor were included in the calculations of ethyl maltol and maltol excreted.


Urinary Excretion.
Determination of free ethyl maltol
Background and recovery. Four normal urine samples (20 ml each) were adjusted to pH 5.0, extracted with five times 20 ml of chloroform, the extract was evaporated in vacuo at 45" C, the residue dissolved in 5 ml of methanol, 1 ml of ferric ammonium sulfate reagent added, and diluted to 10 ml with water. The absorbance was measured at 520 nm against a reagent blank. The mean value for background was found to be 0.1. Recovery (normal urine fortified with ethyl maltol) amounted to 98%. Free ethyl maltol was determined by extraction of urine samples of dosed animals as described above.

Determination of total conjugates
2. 0 ml of urine of each dog of each of the two subsequent 24 hr collections were hydrolyzed by refluxing with equal volumes of 6N sulfuric acid for 2 hr. Extractions and assays were performed as described.

Separate determination of sulphate and glucuronide
An aliquot of the urine samples was mixed with the necessary amount of 2Nsulfuric acid to give a 0.25Nacid solution. The mixture was heated in a steam bath for 15 min, cooled, and extracted with five times 20 ml of chloroform. The combined chloroform extracts were processed and assayed as described. The aqueous phase from the chloroform extraction was mixed with an equal volume of 6N sulfuric acid and refluxed for 2 hr. The hydrolyzate was processed as described above.

Results and discussion

Main ADME results

Main ADME results 1

Type:

metabolism

Results:

An average of 58.5% of the administered dose was excreted as a mixture of sulfate and glucuronic acid conjugates of maltol at 72hrs.

Main ADME results 2

Type:

excretion

Results:

About 98% of the total urinary excretion of conjugates occurred within the first 24 h, males and females excreting an average of 42% and 73% of the administered dose, respectively.

Toxicokinetic / pharmacokinetic studies

Details on excretion:

About 98% of the total urinary excretion of conjugates occurred within the first 24 h, males and females excreting an average of 42% and 73% of the administered dose, respectively (Table 2).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

An average of 58.5% of the administered dose was excreted as a mixture of sulfate and glucuronic acid conjugates of maltol (Table 2).

Applicant's summary and conclusion

Conclusions:

In the Beagle dog, Maltol are predominantly metabolized to sulfate and glucuronic acid conjugates, which are then eliminated in the urine.

Executive summary:

In a metabolism/excretion crossover design study (Rennhard, 1971), Maltol was administered to 2 Beagle dogs of each sex via IV injection at a dose level of 10 mg/kg bw. Urine samples were collected for 3, 6, 24, 48 and 72 hr. The second administration was done 7 days after the first administration. The cumulative urinary excretion of Maltol conjugates (sulfate and glucuronide) in percent of the administered dose was reported.

An average of 58.5% of the administered dose was excreted as a mixture of sulfate and glucuronic acid conjugates of Maltol. About 98% of the total urinary excretion of conjugates occurred within the first 24 h, males and females excreting an average of 42% and 73% of the administered dose, respectively.