Ethyl enantate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The relative rate of hydrolysis by pancreatic lipase of the test item was determined in vitro.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Not applicable

Route of administration:

other: not applicable; in vitro experiment

Vehicle:

other: not applicable; in vitro experiment

Details on exposure:

not applicable

Duration and frequency of treatment / exposure:

not applicable

No. of animals per sex per dose / concentration:

not applicable

Control animals:

other: not applicable

Details on study design:

STARTING MATERIAL
- Substrate: esters of primary n-alcohols containing 1-18 carbons and fatty acids containing 2-18 carbons
- Fatty acids: isolated from natural fats or purchased
- Alcohols: obtained from commercial sources
- Esters: synthesized from the fatty acids and alcohols, and purified by appropriate distillation, crystallization, and column chromatography.

ENZYME
- Preparation: lipolytic enzymes, other than lipase, from rat pancreatic juice were freeze-dried and inactivated for 1 hour
- pH: 9
- Temperature: 40°C

DIGESTION
- Mixture: 225 µmoles of substrate, 330 µmoles CaCl2, 7 µmoles free oleic acid, 17 mg histidine (final concentration 0.002 M), 3.11 g NaCl (final concentration 1 M) and 0.6 mg selectively inactivated, lyophilized rat pancreatic juice in a total volume of 55 ml
- pH: 9.0
- Temperature: 25°C

DATA EVALUATION
The activity of the enzyme preparation varied slightly from day to day. To correct this, replicate samples of methyl oleate were hydrolyzed each day. The values for the other esters were corrected to the standard value for the methyl oleate.

Statistics:

The values for the rates of hydrolysis of the esters are for the first few minutes after addition of the enzyme.
10% difference between two rates is significant.

Type:

metabolism

Metabolites identified:

not specified

Rat pancreatic lipase hydrolyses the test item at 1.4 µeq/min/mg enzyme.

Conclusions:

Rat pancreatic lipase hydrolyses the test item at a fast rate. The relative hydrolysis rate of the test item by the pancreatic lipase was 1.4 µeq/min/mg enzyme.

Executive summary:

In the current study the rate of hydrolysis of the test item by rat pancreatic lipase was determined. In the study various esters of primary n-alcohols, containing 1 to 18 carbon atoms with fatty acids containing from 2 to 18 carbon atoms, were analysed. The test item - ethyl heptanoate - is one of the substrates examined in this experiment.

For the experimental conditions, the concentration of the substrate exceeded that of its solubility in water to assure an interface. The solidification point of the substrate was lower that the temperature of digestion to ensure a liquid/liquid interface.

The objective of the study was to determine the relative hydrolysis rates of esters, both short- as long-chained. Esters are hydrolyzed to an alcohol and a fatty acid. During the course of the study it was observed that the long-fatty acids had an accelerating effect on the hydrolysis, probably due to substrate orientation.

Rat pancreatic lipase hydrolyses the test item at a fast rate. The relative hydrolysis rate of the test item by the pancreatic lipase was 1.4 µeq/min/mg enzyme.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The hydrolysis rate of the test item by artificial gastrointestinal juices and fresh preparations of rat liver and small intestine was determined.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

- Body weight: 200 - 250 g
- Diet: Spillers Laboratory Animal Diet
- Water: ad libitum

Sex:

male

Route of administration:

other: not applicable

Vehicle:

other: not applicable

Details on exposure:

The animals were not exposed to the test item. Fresh preparations of the liver and the small intestine of the animals were made.

Duration and frequency of treatment / exposure:

not applicable

No. of animals per sex per dose / concentration:

unknown

Positive control reference chemical:

not applicable

Details on dosing and sampling:

not apllicable

Preliminary studies:

The solubility at 37°C was 17 mg/100mL water.
The esterase activity of the artificial juices and rat tissues for the test item were:
- Artifical gastric juice: 0.18 mg ester hydrolysed/L/min
- Artifical pancreatic juice: 13.9 mg ester hydrolysed/L/min
- Liver: 49.7 mg ester hydrolysed/g wet tissue/min
- Small intestinal mucosa: 14.9 mg ester hydrolysed/g wet tissue/min

Type:

metabolism

Ester hydrolysis by artificial gastrointestinal juices:

- Artifical gastric juice: The value of the ester hydrolysis constant was 0.054 K/h and the t_1/2 was 770 min.

- Artifical pancreatic juice: The value of the ester hydrolysis constant was 4.25 K/h and the t_1/2 was 9.78 min.

Ester hydrolysis by tissue preparations:

- Liver: The value of the ester hydrolysis constant was 15,200 K/h and the t_1/2 was 1.64 E-1 secondes

- Small intestinal mucosa: The value of the ester hydrolysis constant was 4540 K/h and the t_1/2 was 5.50 E-1 secondes.

Conclusions:

The test item is raplidly hydrolysed by fresh preparations of liver and small intestinal mucosa of the rat.

Executive summary:

In the current study the hydrolysis rates of the test item was determined by artificial gastrointestinal juices and fresh preparations of rat liver and small intestine. The test item was one of sixteen esters that were evaluated.

The solubility of the test item in water was determined to be 17 mg/ 100 mL water.

For the hydrolysis by artificial gastrointestinal juices, the artificial gastric and pancreatic juices were incubated with 17 mg/100mL water of the test item and at intervals, portions of the solution were added to a 0.16% solution of standard in solvent (internal standard: p-Cresol methyl ether), shaken, and left on ice to separate. Samples were withdrawn from the hydrolysis flask at 0.5, 1, 2 and 4 h, and analysed with gas chromatograph.

For the fresh preparations animals were killed by cervical dislocation and 10% homogenates of the liver and the small intestine were prepared, where after the hydrolysis by tissue homogenates was immediately started. Similar as for the artificial gastrointestinal juices 17 mg of the test item/100mL water was incubated with the fresh tissue preparations and for extraction p-Cresol methyl ether was used. Samples were withdrawn from the hydrolysis flask at 0, 10, 20 and 30 min for liver homogenates and 0, 3, 6 and 10 min for intestinal mucosal preparations, and analysed with gas chromatograph.

The test item was hydrolysed relatively slowly by artificial gastric juice, while the reaction with artificial pancreatic juice was faster.

Regarding the fresh preparations the ester was hydrolysed with greater facility than by the artificial pancreatic juice. Both the rat liver homogenates and the intestinal mucosal preparations were found to more efficient than artificial pancreatic juice.

The artificial gastrointestinal juices exhibited a limited ability to hydrolyse the test item, while rat liver and small intestinal preparations were found readily to hydrolyse the test item to the acid and alcohol components. These findings show a large difference between the results obtained from the two methods. The use of tissue preparations is likely to present a more biological relevant toxicological assessment.

Description of key information

The test item is rapidly hydrolysed by pancreatic lipase, rat liver homogenate and rat small intestine homogenate.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Absorption

No studies are available that address the absorption of the test substance via the oral, dermal or inhalation route. Nevertheless, some preliminary predictions can be made from its physico-chemical properties.

Oral absorption

Oral absorption can occur along the entire length of the gastro-intestinal tract. However, it should be kept in mind that substances can undergo chemical changes in the gastro-intestinal fluids prior to absorption. This is of particular relevance for ethyl heptanoate, as further explained below in the section on metabolism. Ethyl heptanoate will undergo an enzymatic hydrolysis of its ester moiety under the influence of gastro-intestinal esterase enzymes. This hydrolysis process gives rise to the formation of ethanol and heptanoic acid, both of which can subsequently be absorbed from the gastro-intestinal tract.

Dermal absorption

A number of physico-chemical factors determine the extent to which a substance may be absorbed by the dermal route. In order for a substance to cross the stratum corneum, the substance should be sufficiently lipophilic (= sufficient solubility in fat). On the other hand, partitioning from the stratum corneum into the epidermis requires sufficient hydrophilicity (= sufficient solubility in water). Hence, the likelihood of dermal absorption is determined by the substance's log Kow and water solubility values.

According to Nielsen et al. (2010), dermal absorption is likely for a substance if it has the following properties:

- a vapour pressure < 100 Pa

- a log Kow between 1 and 4

- a water solubility in the range of 1 - 100 mg/L (moderate absorption), or 100 - 10000 mg/L (high absorption).

Ethyl heptanoate has a vapour pressure of 427 Pa, a log Kow of 3.98 and a water solubility of 126 mg/L. Based on these physicochemical properties, it can be concluded that the dermal absorption of the substance can be considered to be likely, i.e. the substance is sufficiently soluble in both fat and water to allow for dermal uptake. However, the volatility of the substance will to a certain extent limit its dermal uptake, as evaporation from the skin will also take place.

Inhalation absorption

The main physicochemical parameters determining the extent to which a liquid substance may be absorbed by the inhalation route are the vapour pressure, the log Kow and the water solubility.

Nielsen et al. (2010) gives the following criteria:

- Highly volatile substances are considered those substances that have a vapour pressure exceeding 25000 Pa (or boiling point < 50°C), whereas a vapour pressure < 500 Pa (or boiling point > 150C°) indicates a low volatility.

- Log Kow > 0 indicates the potential for direct absorption across the respiratory tract epithelium. Substances with a log Kow value between 0 and 4 are sufficiently lipophilic to allow crossing the alveolar and capillary membranes, and hence are likely to be absorbed as well.

- A sufficient water solubility increases the potential for inhalation absorption. Nevertheless, very hydrophilic substances may be retained within the mucus, and transported out of the respiratory tract by clearance mechanisms.

Based on the physicochemical properties of ethyl heptanoate (Log Kow 3.98 and water solubility 126 mg/L), it can be concluded that absorption of the substance following exposure via the inhalation route is likely. However, the relatively low vapour pressure (427 Pa) and high boiling point (187.5°C) will to a certain extent mitigate the likelihood of exposure via the inhalation route.

Metabolism and Excretion

As mentioned above, the metabolism of ethyl heptanoate is determined by the substance's tendency to undergo enzymatic hydrolysis, giving rise to the formation of ethanol and heptanoic acid.

Primary metabolic step: enzymatic hydrolysis of ethyl heptanoate

Regarding this enzymatic hydrolysis, there are 2 studies available in which the enzymatic hydrolysis rate of the substance was assessed.

In the study of Mattson and Volpenhein (1969) the in vitro hydrolysis of a large set of primary esters - including ethyl heptanoate - was assessed. In this study the substrate was added in excess (i.e. exceeding its water solubility) to a digestion mixture containing an enzyme preparation obtained from rat pancreatic juice. The experiment showed that the rat pancreatic lipase hydrolysed the test item at a fast rate. The relative hydrolysis rate of the test item by the pancreatic lipase was 1.4 µeq/min/mg enzyme.

In the second study (Longland, 1977) the hydrolysis rates of artificial gastrointestinal juices and fresh prepared rat liver and small intestine homogenates were investigated.

The test item was added in excess (exceeding its water solubility) and after certain time intervals a sample was taken and analysed by gas chromatography. The time intervals were as follows:

- artificial gastrointestinal juices: 0.5, 1, 2 and 4 h

- liver homogenates: 0, 10, 20 and 30 min

- intestinal mucosal preparations: 0, 3, 6 and 10 min

All preparations were able to hydrolyse the test item.

The test item was hydrolysed relatively slowly by artificial gastric and pancreatic juices in comparison with the freshly prepared rat liver homogenates and the intestinal mucosal preparations. The rat liver and small intestinal preparations were found to rapidly hydrolyse the test item.

Further metabolism and excretion of the primary metabolites

The primary metabolites formed upon the ezymatic hydrolysis of ethyl heptanoate are ethanol and heptanoic acid.

The metabolism of ethanol has been studied in great detail due to its presence in alcoholic beverages. The main (though not only) metabolic pathway for ethanol is conversion to acetaldehyde by means of alcohol dehydrogenase, and subsequent conversion into acetate by means of aldehyde dehydrogenase. The acetate is further broken down into CO₂ and water.

Heptanoic acid is a saturated carboxylic acid. Such substances are metabolized in the fatty acid beta-oxidation pathway and the citric acid cycle (Krebs cycle). In the fatty acid beta-oxidation pathway, the carboxylic acids are condensed with coenzyme A (CoA) to the corresponding acyl CoA thioesters. The thioester undergoes beta-cleavage, which gives rise to the formation of acetyl CoA and a new acyl-CoA thioester which is 2 carbon atoms shorter than the original acyl CoA thioester. The cycle then continues, shortening the chain of the carboxylic acid moiety until ultimately acetyl CoA (for even-numbered carboxylic acids) or propionyl CoA (for odd-numbered carboxylic acids) is formed. Acetyl CoA enters the citric acid cycle directly, whereas priopioinyl CoA is converted to succinyl CoA before, in turn, entering the citric acid cycle (WHO 1998). In the citric acid cycle, the CoA is released again and the acetyl and succinyl groups are oxidised to CO₂.

Ultimately, the final metabolite of both ethanol and heptanoic acid is CO₂, which is excreted via the respiratory route.

References:

Nielsen E, Ostergaard G, Larsen JC (2010). Toxicological Risk Assessment of Chemicals; A Practical Guide. Published by: Informa Healthcare. ISBN-13: 9780849372650.

WHO (1998). WHO Food Additives Series: 40. No. 905: Substances evaluated using the procedure for the safety evaluation of flavouring agents. WHO, Geneva, 1998. International Programme on Chemical Safety.