Methyl cinnamate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented, published in peer-reviewed literature, performed to non-standard protocol, but contributing to assessment.

Data source

Reference

Reference Type:

publication

Title:

The absorption and metabolism of methyl cinnamate.

Author:

Fahelbum and James,

Year:

1977

Bibliographic source:

Toxicology, 7, 123-132.

Report date:

1976

Materials and methods

Objective of study:

other: absorption and metabolism

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

not applicable

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl cinnamate

EC Number:

203-093-8

EC Name:

Methyl cinnamate

Cas Number:

103-26-4

Molecular formula:

C10H10O2

IUPAC Name:

methyl 3-phenylacrylate

Test material form:

not specified

Radiolabelling:

no

Test animals

Species:

other: rabbit and rat

Strain:

other: rabbit: White New Zealand; rat: Birmingham Wistar

Sex:

not specified

Details on test animals or test system and environmental conditions:

Animals
Doe rabbits (White New Zealand strain, 3—4 kg) maintained on Oxoid Diet 18 and female rats (Birmingham Wistar strain, 250—400 g), on the Hey gates modified rat-mouse breeding diet, were used. The animals were housed in metabolism cages which permitted the separate collection of urine and faeces. Urine was collected for 24 h before dosing and for successive 24 h periods afterwards. In investigations of the absorption of compounds from the gut the rats were allowed free access to food before dosing and were not fed thereafter. Cinnamic acid was administered as a suspension in warm water (37 °C) and methyl cinnamate as a melted suspension in water.
In an attempt to establish the major site of absorption of the ester, in some experiments the stomach of a rat anaesthetised by intraperitoneal injection of Nembutal was ligated at the pylorus. Methyl cinnamate was then administered by stomach tube as an aqueous suspension containing bile salts.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

Determination of blood levels of methyl cinnamate and cinnamic acid in animals dosed with methyl cinnamate:
Rabbit: Methyl cinnamate (1.0 mmole/kg) was administered to the rabbits by stomach tube and at intervals after the dose blood samples (10 ml) were taken from the ear vein. Rat: Rats were similarly dosed with methyl cinnamate (1.5 mmole/kg).
Determination of absorption of methyl cinnamate and cinnamic acid:
Rats dosed with cinnamic acid (20 mg) or methyl cinnamate (20 mg) were killed at intervals after the dose.

Duration and frequency of treatment / exposure:

determination of absorption: from 5 to 240 minutes;
determination of blood level: from 15 to 300 minutes;
determination of excretion: 24h

Doses / concentrations

Remarks:

Doses / Concentrations:
determination of absorption: 20mg;
determination of blood level: rabbit: 3.1mmole; rat: 0.62 mmole;
determination of excretion: rabbit 500mg; rat: 50 mg
rabbit: 1.0 mmole/kg;
rat: 1.5 mmole/kg

No. of animals per sex per dose / concentration:

N/A

Control animals:

not specified

Positive control reference chemical:

N/A

Details on study design:

Determination of absorption of methyl cinnamate and cinnamic acid
Determination of blood levels of methyl cinnamate, methanol and cinnamic acid in animals dosed with methyl cinnamate. (a) Determination of methyl cinnamate and cinnamic acid
Determination of methanol in blood,
Enzymic hydrolysis of methyl cinnamate: (a) In blood; (b) By rat liver homogenate; (c) By gut scrapings.
Identification and determination of metabolites excreted in urine

Details on dosing and sampling:

Determination of absorption of methyl cinnamate and cinnamic acid
Rats dosed with cinnamic acid (20 mg) or methyl cinnamate (20 mg) were killed at intervals after the dose by a blow on the head and the gut was care- fully removed and dissected into four sections, stomach, small intestine, caecum and large intestine. Each section was homogenised in water (20 ml), the homogenate acidified to pH 1-2 by the addition of hydrochloric acid and continuously extracted with ether for 6 h. The extracts from the gut sections of animals dosed with cinnamic acid were esterified by the addition of an ethereal solution of diazomethane. The extracts were evaporated to dryness and dissolved in methanol (1—8 ml). The extracts from the gut sections of animals dosed with methyl cinnamate were evaporated and then dissolved in methanol. Half of the solution was retained, without esterification, for analysis to measure intact ester and half was treated with diazomethane to esterify any acid arising from the dosed ester. A known amount of a solution of methyl hexylmercapturate was added to the extracts as the internal standard; duplicate aliquots of the solution were analysed by GLC using a Pye series-104 dual-flame ionisation chromatograph fitted with columns packed with silanized chromosorb-W coated with 5% QF-1. The carrier gas was argon and the flow rate 45 ml per min. The temperature was raised from 100 °C to 240 °C at a rate of 8 °C per min. The areas of the peaks corresponding to the esters and the internal standards were measured. Calibration curves prepared for methyl cinnamate and methyl hexylmercaptu- rate were used to calculate the amount of ester in the samples examined. The mean percentage recoveries by this method of cinnamic acid and methyl cinnamate added to homogenised gut, with the and the number of determinations in brackets were: cinnamic acid 90.1 ±2,0 (5); methyl cinnamate before esterification 86.8 ±1.2 (5); after esterification 89.4 ± 1.0 (5). A comparison of the recovery figures for the ester before and after esterification indicate that less than 3% of the ester is hydrolysed under the conditions used.
In the determination of the unchanged ester in the gut the recovery figure for the ester “without esterification” was used. For the determination of total acid plus esttir the recovery for ester after esterification was employed.
Determination of blood levels of methyl cinnamate, methanol and cinnamic acid in animals dosed with methyl cinnamate. (a) Determination of methyl cinnamate and cinnamic acid
Rabbit: Methyl cinnamate (1.0 mmole/kg) was administered to the rabbits by stomach tube and at intervals after the dose blood samples (10 ml) were taken from the ear vein. The blood was acidified to pH 1-2 by the addition of hydrochloric acid and then continuously extracted with ether for 18 h. The extracts were analysed before and after methylation as described above.
Rat: Rats were similarly dosed with methyl cinnamate (1.5 mmole/kg). At intervals after the dose the rats were anaesthetised with Nembutal (Abbot) and a sample of blood (1-2 ml) taken from the portal vein and a further sample from the heart. Similarly dosed rats were used to obtain successive samples (1 ml) of blood from the tail vein at intervals after the dose; not more than three samples were taken from any one rat. The blood samples were extracted and the acid and ester present determined.
The percentage recoveries, with the S.E.M. and the number of determinations, of methyl cinnamate and cinnamic acid added to blood were: cinnamic acid 87.7 ± 2.3; methyl cinnamate 88.0 ±1.6 (before esterification); 91.0 ± 1.8 after esterification. The method permitted the detection in blood of 0.014 /zmole/ml of methyl cinnamate.
(b) Determination of methanol in blood
Blood samples (1-2 ml) taken as described above were deproteinised by the addition of 10% (w/v) zinc sulphate solution (0.4 ml) followed by 0.5 M NaOH (0.5 ml). A measured amount of propanol was added as the internal standard and the solutions were well mixed and then centrifuged. Aliquots of the supernatant were applied to a column packed with Poropak Type Q (Waters Associates Inc., Mass.) maintained at 170° C in the gas chromatograph previously described. Under these conditions the retention times for methanol and n-propanol were 1.45 and 5.9 min, respectively. The recovery of methanol added to blood was 89.3 ± 3.1 (5) and the method detected 0.1 µmole/ml.
Enzymic hydrolysis of methyl cinnamate
(a) In blood. Samples of rabbit blood or serum (5 ml) were incubated at 37 °C with methyl cinnamate (62µmmoles) and rat blood or serum (2 ml) were similarly incubated with the ester (31 µmoles). Ethereal extracts of the acidified digests were analysed for ester and ester plus acid as previously described. No significant hydrolysis of the ester occurred in control experiments.
(b) By rat liver homogenate. Rat liver was homogenised in 6 vol. of 0,1 M phosphate buffer pH 7.4 and aliquots (5 ml) were incubated with methyl cinnamate (62 µmoles). The hydrolysis was followed as previously described either by measuring the ester and cinnamic acid present in samples withdrawn from the digest or by determination of the methanol present in deproteinised samples. A control in which methanol (60 µmoles) was incubated with liver homogenate (5 ml) was also examined.
In a further series of experiments the Km and Vmax values for the hydrolysis of methyl cinnamate were determined by the method described by Wynne et al. in which the protons released on hydrolysis are titrated with 0,02 M NaOH. For this a Titrigraph pH stat (Radiometer Copenhagen) Type TTT lc connected with an SBR 2c recorder and Syringe Burette (Type SBU K) with temperature compensator PHA 924 was used The digest, vigorously mixed by means of a magnetic stirrer, consisted of 0.1 M NaCl (2.5 ml), acetonitrile or a solution of the ester in acetonitrile (0.03 ml) and the liver preparation (0.5 ml). The latter consisted of the supernatant obtained by centrifugation of a 10% (w/v) homogenate of liver in 0.1 M NaCl for 30 min at 1000 g.
(c) By gut scrapings. In duplicate experiments two rats were killed by a blow on the head and their stomachs and small intestines separately removed. These "were opened and washed with a gentle stream of water. The linings of the stom- achs and of intestines were carefully scraped off and the stomach linings from "both animals were homogenised in phosphate buffer pH 7.4 (10 ml). The linings of the small intestines were similarly treated. Methyl cinnamate (61 µmoles) was added to each homogenate which was incubated at 37 ° C for 20 min. The amount of ester and acid present in each homogenate was then determined by g.l.c. The protein content of the homogenates was determined.
Identification and determination of metabolites excreted in urine
The metabolites excreted by rabbits and rats which had been dosed with methyl cinnamate or cinnamic acid were identified by paper and t.l.c. of samples of urine or of ethereal extracts of acidified urine and by g.l.c of samples of the ethereal extracts which had been esterified by the addition of diazomethane. Hippuric acid excretion was measured by g.l.c. and by the method of Gaffney et al，,glucosiduronic acids as described by Bray et al. except that the colours were read on an EEL colorimeter using filter 601.

Statistics:

N/A

Results and discussion

Preliminary studies:

N/A

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The results show that the major fraction of the unabsorbed dose was present in the stomach and that the rates at which the ester and acid disappeared from the gut were closely similar. And little hydrolysis of the ester occurs in the stomach lumen.

Main ADME results 2

Type:

distribution

Results:

The levels of cinnamic acid rose gradually in both species reaching a maximum at 2 h after the ester was administered falling gradually thereafter and after 5 h was 0.11 µmole/ml in rabbit peripheral blood and after 4 h 0.17 µmole/ ml in that of the rat.

Main ADME results 3

Type:

metabolism

Results:

metabolites: cinnamic acid and methanol, p-hydroxyhippuric acid by rabbits dosed with cinnamic acid or methyl cinnamate

Main ADME results 4

Type:

excretion

Results:

via urine

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Analysis of the gut contents of rats killed at intervals after dosage with methyl cinnamate or cinnamic acid suggested that both ester and acid were rapidly absorbed; at no time was more than 5% of the dose detected in the lower part of the gut.
Absorption from the gut
The percentages of the dose of unabsorbed cinnamic acid and methyl cinnamate present in the different sections of the gut are given in Table I. The results show that the major fraction of the unabsorbed dose was present in the stomach and that the rates at which the ester and acid disappeared from the gut were closely similar. Up to 2 h after its administration the dose present in the stomach represented an average of 88—97% of the unabsorbed material present in the whole gut for both the ester and acid. In this same period up to 23% of the dose of methyl cinnamate remaining in the stomach had been hydrolysed; methyl cinnamate was not detected in the caecum or large intestine and the amount of unhydrolysed ester present in the small intestine did not exceed 2.1% of the dose. The pKa value for cinnamic acid is 4.44 so that at the pH of the rat stomach (found in this study to be 3.95) 68% of cinnamic acid will be undissociated. The acids would therefore be expected to be readily absorbed (Schanker et al.), The lipid solubility of the ester might be expected to promote its absorption but the results did not suggest that the ester was absorbed at a rate significantly greater than that of the parent acid.
In experiments using rats in which the stomach was ligated at the pylorus in one animal 44.5% of the methyl cinnamate (22 mpr) administered orally was present in the stomach after 2 h and with two other animals receiving doses of 36 mg the corresponding figures were 26 and 32%, In the rat receiving the lower dose the cinnamic acid present amounted to 0.9% of the dose the corresponding figure for the higher dose level being 1.9% in both cases. These results show that under these conditions little hydrolysis of the ester occurs in the stomach lumen. While they indicate that absorption occurs from the stomach the possibility is not excluded that in the intact ani¬mal a proportion of the dosed ester passes into the duodenum from where it is rapidly absorbed.

Details on distribution in tissues:

Not more than 9% of the administered methyl cinnamate was detected in the stomach as cinnamic acid whereas at least 40% of the small amounts of the dosed eater detected in the lower part of the gut was present as cinnamic acid. No ester was detected in the peripheral blood of dosed rabbits or rats and only traces were detected in portal and heart blood samples taken from dosed rats.
Blood levels of methyl cinnamate, cinnamic acid and methanol after the ad-ministration of the ester
The administered ester was not detected in any of the samples of peripheral blood of either rabbit or rat taken from 15 to 300 min after the dose was given. The levels of cinnamic acid rose gradually in both species reaching a maximum at 2 h after the ester was administered falling gradually thereafter and after 5 h was 0.11 µmole/ml in rabbit peripheral blood and after 4 h 0.17 µmole/ ml in that of the rat.
The concentration of methanol in the peripheral blood of rabbits and rats which had been dosed with methyl cinnamate is recorded in Table II. In the rat methanol concentration reached a higher level than that of cinnamic acid in animals which had received the same dose of the ester and the level fell more slowly than that of cinnamic acid. This difference was not apparent in the rabbit which received a lower dose of the ester.
The concentration of methyl cinnamate and cinnamic acid determined in samples of portal and heart blood taken from rats which had been dosed with methyl cinnamate are also given in Table II. Not more than a trace of the intact ester was detected in samples of blood taken from the heart while the amount of intact ester in portal blood did not amount to more than 12% of the total ester plus acid present and none was detected in most of the samples analysed.

Details on excretion:

In addition to the metabolites of cinnamic acid described in the literature p-hydroxyhippuric acid was excreted as a minor metabolite of both cinnamic acid and methyl cinnamate.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Cinnamic acid and methanol were readily detected in the blood of rabbits and rats which had been dosed with methyl cinnamate. No qualitative or significant quantitative difference was detected in the metabolism of the ester as compared with the parent acid. In addition to the metabolites of cinnamic acid described in the literature p-hydroxyhippuric acid was excreted as a minor metabolite of both cinnamic acid and methyl cinnamate.
rabbit: Metabolites were isolated and examined by paper, TLC (thin layer chromatography) and GLC (gas–liquid chromatography). The following metabolites were identified as a percentage of the dose: hippuric acid (56.0%) and glucosiduronic acid (8%).
rat: The following metabolites were identified as a percentage of the dose: hippuric acid (67.0%) and glucosiduronic acid (3%).

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

N/A

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
It is concluded that test article can be absorbed rapidly and distributed in the blood. The metabolites of test article was identified to be cinnamic acid and methanol which sequently been metabolized to p- hydroxyhippuric acid, excreting as a minor metabolite of test article.

Executive summary:

This paper was published to investigate the toxicokinetics information of test article.

Analysis of the gut contents of rats killed at intervals after dosage with methyl cinnamate or cinnamic acid suggested that both ester and acid were rapidly absorbed; at no time was more than 5% of the dose detected in the lower part of the gut. Not more than 9% of the administered methyl cinnamate was detected in the stomach as cinnamic acid whereas at least 40% of the small amounts of the dosed eater detected in the lower part of the gut was present as cinnamic acid. No ester was detected in the peripheral blood of dosed rabbits or rats and only traces were detected in portal and heart blood samples taken from dosed rats. Cinnamic acid and methanol were readily detected in the blood of rabbits and rats which had been dosed with methyl cinnamate. No qualitative or significant quantitative difference was detected in the metabolism of the ester as compared with the parent acid. In addition to the metabolites of cinnamic acid described in the literature p-hydroxyhippuric acid was excreted as a minor metabolite of both cinnamic acid and methyl cinnamate.