Phenethyl acetate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The potential for hydrolysis of a series of esters widely used as flavouring agents in foods and with uses in cosmetics and plastics/paint manufacture was evaluated using artificial gastrointestinal juice and liver and small intestine tissue freshly prepared from Wistar rats.

GLP compliance:

not specified

Test material

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

The solubility of the esters in water was determined by shaking a known weight of ester, in excess of the quantity soluble in 100 ml water, with 100 ml water for 30 min at 37°C, and allowing to separate at that temperature. From the saturated solution 50 ml was withdrawn and this,and the other 50 ml of saturated solution together with the undissolved ester, was separately extracted with 10 ml of the appropriate solvent and standard solution. The amount of ester in each extract was determined by gas-liquid chromatography, and the solubility of ester in water at 37°C was calculated.

Administration / exposure

Route of administration:

other:

Vehicle:

not specified

Details on exposure:

For assessment of hydrolysis by artificial gastrointestinal juices:
The artificial gastric and pancreatic juices were prepared as described in Pharmacopoeia Helvetica VI, modified in the case of the pancreatic juice by increasing the ionic strength of the phosphate buffer to maintain the pH at 7.5 during the hydrolysis of the esters. The artificial gastric juice consisted of: sodium chloride 2.0 g, in 1 N HC180.0 ml and pepsin 3.2 g made up to one litre with distilled water and adjusted to pH 1.2. The artificial pancreatic juice consisted of: pancreatin 10.0 g, sodium taurocholate 0.5 g, disodium hydrogen phosphate 50.5 g and sodium dihydrogen phosphate dihydrate
15.6 g, made up to one litre with distilled water and adjusted to pH 7.50.
The artifical juice (110 ml) was placed in a stoppered 250-ml flask in a shaking incubator at 37°C. A measured ,quantity of ester, a little less than that required to give a saturated solution, was added, the flask shaken vigorously for 2 min and returned to the incubator. At intervals, 20-ml portions· of solution were pipetted into 25-ml stoppered flasks containing
5 ml of 0.16% solution of standard in solvent, cooled in ice, shaken vigorously for 2 min and left on ice to separate. As soon as sufficient solvent phase had separated, a sample was injected into the gas chromatograph. Generally, samples were withdrawn from the hydrolysis flask at 0.5, 1, 2 and 4 h, but the intervals were decreased when the ester was found to hydrolyse quickly.
For hydrolysis by tissue homogenates
The animals were killed by cervical dislocation, the abdomen opened and the liver and approx. 10-cm length of small intestine within the region between 10 and 30 em from the pylorus removed. Liver homogenate (10%) was prepared in ice-cold 1.15% KGI 1 mM EDTA buffer pH 7.4. The small intestine was flushed through with ice-cold Krebs-Ringer solution, slit along its length and the mucosal surface removed. Mucosal homogenate (10%) was prepared in ice-cold oxygenated Krebs-Ringer solution containing glucose (200 mg/100 ml). Hydrolysis studies were commenced immediately after the preparation of the tissue homogenates. The procedure used was similar to that described for the artificial gastrointestinal juices except that portions of samples were removed from the reaction flasks at shorter time intervals; 0, ~O, 20 and 30 min for liver homogenates and 0, 3, 6 and 10 min .for intestinal mucosal preparations. Additionally, when ester hydrolysis was found to proceed extremely rapidly, the tissue assays were conducted with diluted tissue preparations.
The 5-ml aliquots removed from the reactions flasks were delivered into ice-cold tubes containing 1.5 g of NaGI, methanol (10 ml) and chloroform (5 ml). After shaking the tubes vigorously for 2 min, 5 ml of chloroform containing the internal standard was added and the tube further shaken for 30 sec, 5 ml of saturated NaGI solution was then added and tne shaking continued
for 30 sec. The tube was centrifuged at 5000 rev./min for 10 min and the chloroform layer removed for gas chromatography.
Gas-liquid chromatographic analysis was conducted on a Pye series 104 dual flame ionization chromatograph. The columns were 5 feet X 4 mm glass, packed with 100/120 mesh celite impregnateCl with 10% Garbowax 20 M or with polyethylene glycol adipate. The chromatographs were run isothermally, using nitrogen at 50 ml/min as the carrier gas.

Duration and frequency of treatment / exposure:

5 ml of 0.16% solution of standard in solvent, cooled in ice, shaken vigorously for 2 min and left on ice to separate. As soon as sufficient solvent phase had separated, a sample was injected into the gas chromatograph. Generally, samples were withdrawn from the hydrolysis flask at 0.5, 1, 2 and 4 h, but the intervals were decreased when the ester was found to hydrolyse quickly.

No. of animals per sex per dose / concentration:

Not applicable

Control animals:

no

Positive control reference chemical:

Not applicable

Details on study design:

No further details

Details on dosing and sampling:

Not applicable

Statistics:

Not applicable

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolite characterisation studies

Details on metabolites:

Not applicable

Any other information on results incl. tables

Ester hydrolysis by artificial gastrointestinal juices followed first-order kinetics. Values for the ester hydrolysis rate constant (K) and t0.5 (time to effect 50% hydrolysis) showed a relatively slow rate of hydrolysis by artificial gastric juice and a faster reaction in artificial pancreatic juice. Based on velocity of ester hydrolysis in pancreatic juice the esters were broadly placed into one of three categories - t0.5 <10 mins; t0.5 <45 mins and t0.5 >>45 mins. Phenylethyl acetate was in the middle group. The hydrolysis rate constant (K/h) was 0.139 and t0.5 = 300 mins in gastric juice and in pancreatic juice the values were 1.4 and 29.7 respectively.

In the hydrolysis studies with rat tissue preparations - liver and small intestinal tissue - weight of the esters were selected for analysis, representing the three groupings identified above. Benzyl isobutyrate and isoamyl butyrate were the surrogates for phenylethyl acetate selected to represent Group 2. The rate constant (K/h) in liver tissue was 5070 -59100 and t0.55 in the range 4.22 x 10E-1 to 4.92x 10E-2. In intestinal mucosal tissue the respective values were 35000 -35300 and 7.07 to 7.13 x 10E-2. Hydrolysis in tissue preparations followed first order rate kinetics but the tissue rates showed the tissue preparations hydrolysed the esters much more readily than artificial pancreatic juice (8 ->25000 fold better for liver tissue and 30 ->15000 fold better for intestinal mucosal tissue).

The results show relatively rapid hydrolysis of phenylethyl acetate by artificial gastro-intestinal juices and by liver and intestinal tissues and confirm the indications of rapid metabolic detoxification in the liver following repeat exposure and also the lack of bioaccummulation potential.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The results show relatively rapid hydrolysis of phenylethyl acetate by artificial gastro-intestinal juices and by liver and intestinal tissues and confirm the indications of rapid metabolic detoxification in the liver following repeat exposure and also the lack of bioaccummulation potential.

Executive summary:

The rates of hydrolysis for sixteen esters, used as constituents of artifical flavours, were determined in artificial gastrointestinal juices and in fresh preparations of rat liver and small intestine. The artificial gastrointestinal juices exhibited a limited ability to hydrolyse the esters (methyl anthranilate was virtually unaffected). Rat liver and small intestinal preparations were found readily to hydrolyse the esters to their component acids and alcohols. The results show relatively rapid hydrolysis of phenylethyl acetate by artificial gastro-intestinal juices and by liver and intestinal tissues and confirm the indications of rapid metabolic detoxification in the liver following repeat exposure and also the lack of bioaccummulation potential.