Ethyl hexanoate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

Expert statement

Type of information:

other: Evaluation of toxicokinetics based on literature data

Adequacy of study:

key study

Study period:

NA

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Expert statement

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl hexanoate

EC Number:

204-640-3

EC Name:

Ethyl hexanoate

Cas Number:

123-66-0

Molecular formula:

C8H16O2

IUPAC Name:

ethyl hexanoate

Test material form:

liquid

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Oral
Ethyl caproate possesses the phys/chem properties that favor absorption from the GI tract but bioavailability of the parent molecule would be limited because of hydrolysis in the GI tract. As a result, the toxicokinetics of ethyl caproate is largely determined by its rate of hydrolysis in the GI tract and the distribution, metabolism, and elimination of the corresponding alcohol and carboxylic acid hydrolysis products.
According to the ECHA Guidelines (2014) molecules with molecular weights of less than 500 g/mole are small enough to be candidates for absorption by passive diffusion from the GI tract. The molecular weight of ethyl caproate is 144.2 g/mole which would favor its absorption from the GI tract. In addition, ethyl caproate is moderately water soluble (508 mg/L) and has an octanol-water partition coefficient (log Pow) of 2.96. This combination of aqueous and lipid solubility also generally favors absorption by the oral route.
However, ethyl caproate is readily hydrolyzed in the GI tract which would effectively reduce the availability of parent for absorption. Given the relatively short t½ of ethyl caproate in the GI tract (3 minutes) and the GI residence time (24-72 hours in humans) it is reasonable to conclude most of the ingested ethyl caproate will be hydrolyzed before it could be absorbed and therefore consequently would not reach the systemic circulation. Assuming there is an excess of esterases in the GI tract, the hydrolysis of ethyl caproate would obey linear first-order kinetics resulting in 98% of the ingested ethyl caproate undergoing hydrolysis within 20 minutes.
Finally, any intact ethyl caproate that is absorbed would be almost immediately hydrolyzed by esterases in blood. Rapid hydrolysis of isoamyl-3-(2’-furyl) propionate (CAS 7779-67-1) and allyl phenylacetate (CAS 1797-74-6), also small esters, was reported for whole blood preparations. Both esters underwent 98% hydrolysis in guinea pig blood within 1 minute (Pelling, 1980). Further support comes from the in vivo portion of the Pelling study in which guinea-pigs were administered these two esters through a bolus intraduodenal injection and blood levels of intact ester monitored. No intact ester was detected in the blood.
Based on this, the biological effects from ingestion of ethyl caproate would be predicted to result from exposure to the ethanol and the hexanoic acid formed from the hydrolysis of ethyl caproate.
In a combined repeat dose oral gavage toxicity study with reproduction/developmental toxicity screening (SymriseAG, 2017), rats were administered ethyl caproate by oral gavage daily at up to 1000 mg/kg bw/day for at least 50 days. In addition to an increase in salivation noted at the top dose, increased prothrombin time, T4 and kidney weights were also observed in some animals at this dose level and decreased gamma glutamyl transpeptidase was observed in males of all treated groups. These effects were considered not to be of toxicological relevance but they do indicate systemic exposure following oral administration of ethyl caproate.
Although the test item can be expected to be well absorbed, in the absence of experimental oral absorption data a default oral absorption value is used in derivation of DNELs as a worst case scenario. This is based on 100% absorption with a default factor of 2 in the case of oral to inhalation extrapolation giving an oral absoprtion value of 50%.

Dermal
Based on the phys/chem properties, ethyl caproate is likely to be absorbed after dermal application. According to the ECHA Guidelines (2014) molecules with molecular weights of less than 500 g/mole are capable of migration through the skin into systemic circulation. In addition, both water and lipid solubility influences the potential for dermal penetration. These factors have been used in various models for predicting dermal penetration.
The US EPA model (2007) is one of the most widely applied. It utilizes molecular weight and partition coefficient to predict the dermal permeability coefficient according to the following;
log(Kp) = 0.71 log Pow – 0.0061 MW – 2.72

where Kp is expressed in cm/h

The log Pow is 2.96 and the molecular weight is 144.2 g/mol. The resulting dermal permeability coefficient, (Log Kp) is -1.5 cm/hr (Kp = 0.032 cm/hr).

This permeability coefficient can be used to estimate the amount of ethyl caproate absorbed when applied to the skin. The following equation utilizes the dermal permeability coefficient (Kp), the concentration of test substance in water (Cw), the surface area of exposed skin (SA) the exposure time (ET) and a liter to cm3 conversion factor (CF1) to calculate the absorbed dose rate (ADR).

ADR = Cw x SA x ET x Kp x CF1

Solubilization of the test substance is necessary for dermal penetration, even if the substance is applied as a solid. For this calculation, the limit of solubility in water is used. The maximum solubility of ethyl caproate in water is 508 mg/L at 20°C and pH 4.9. Skin pH is 6.5 and therefore slightly acidic so it is considered appropriate to use this solubility value in the derivation. For the surface area 1 cm2 is used to generate a flux value that can be applied across a variety of applications with different surface area values.

For ethyl caproate the ADR is 0.016 mg/cm2/hr or 16 µg/cm2/hr. Thus, dermal exposure to ethyl caproate would be expected to result in systemic exposure. Ethyl caproate has been classified as a skin irritant and this might facilitate dermal penetration. However, once the parent ethyl caproate crosses the stratum cornea, the esterases present in epidermal cells (Jewell et al., 2007) would be expected to hydrolyze the parent to its corresponding alcohol and carboxylic acid.

Since there was no mortality from ethyl caproate in the dermal acute toxicity assay conducted, and no significant clinical observations, it is not possible to corroborate this conclusion with information generated from in vivo studies.

Although the test item can be expected to be well absorbed, in the absence of experimental dermal absorption data a default dermal absorption value is used in derivation of DNELs as a worst case scenario. This is based on the oral absorption with a default factor of 1 in the case of oral to dermal extrapolation giving a dermal absoprtion value of 50%

Inhalation
Ethyl caproate is a liquid at room temperature with reasonable volatility. The vapor pressure of ethyl caproate is high (3 hPa at 20°C). Also if ethyl caproate were aerosolized, absorption across the respiratory epithelium would be likely rapid based on its partition coefficient and small molecular weight. It is also reasonable to expect significant hydrolysis by esterases in the respiratory epithelium (Olson et al., 1993) which would generate ethanol and hexanoic acid as described for the oral and dermal routes of administration. There are no experimental data on the effects of acute or long-term inhalation exposure to the test substance available.

A default inhalation absorption value of 100% is used in derivation of DNELs as a worst case scenario

Details on distribution in tissues:

Distribution
The distribution of ethyl caproate has not been characterized. The systemic effects noted after oral dosing are minimal and not specific enough to determine a distribution of absorbed ethyl caproate. However, as noted, by all routes of exposure, significant hydrolysis of ethyl caproate is expected forming ethanol and hexanoic acid. Both materials are expected to freely distribute systemically and enter intermediary metabolism in all tissues. Therefore, the distribution of ethanol and hexanoic acid would be proportional to organ blood flow. After administration by the oral route, the hydrolysis products would enter hepatic portal circulation resulting in distribution primary to the liver. Dermal absorption would result in general systemic exposure. By inhalation the distribution would also be expected to be more general as absorption by the lungs would result in distribution systemically via cardiac output.
Since the hydrolysis products of ethyl caproate enter into intermediary metabolism it is unlikely that either would bioaccumulate.

Details on excretion:

The excretion kinetics of ethyl caproate are dependent on the hydrolysis of the ester and the metabolic fate of the ethanol and hexanoic acid. For ethanol, the majority of the dose is expected to be metabolized into natural products. The hexanoic acid may be excreted in urine, either as the free acid or conjugated with glucuronic acid. It may also be broken down by fatty-acid metabolism pathways via ß-oxidation and utilized nutritionally.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

As noted above, ethyl caproate is expected to be rapidly and complexly hydrolyzed to ethanol and hexanoic acid either before, or immediately after absorption by all routes of exposure. Both of these hydrolysis products become substrates for intermediary metabolism.

Ethanol (CAS 64-17-5): Ethanol is oxidized by alcohol and aldehyde dehydrogenases to eventually form acetyl CoA which joins the citric acid cycle / natural carbon pool to be converted into CO2, fatty acids, ketone bodies and cholesterol (Cederbaum, 2012). The metabolism of ethanol results in the formation reactive oxygen species, which can, in excess result in cellular oxidative stress if these reactive molecules are present at levels which cannot be dealt with by anti-oxidant species. This process is considered to be a key event in the development of alcohol–induced liver disease. The exposure to ethanol resulting from the use of ethyl caproate is highly unlikely to result in the levels these molecules would need to be present to cause cellular injury.

Hexanoic acid (CAS 142-62-1): This straight chain carboxylic acid with an even number of carbon units is a substrate for endogenous fatty-acid metabolism that occurs via ß-oxidation (Nelson and Cox 2005). Within this system, aliphatic carboxylic acids undergo conjugation with acetyl CoA and oxidation of the bond between the 2nd and 3rd carbons adjacent to the carboxylic acid. This oxidation then leads to the ultimate cleavage at the site of oxidation to release two carbons in the form of acetyl CoA. This process continues in a cyclic fashion two carbons at a time until all of the carbons in the fatty acid are converted to acetyl CoA.

Any other information on results incl. tables

This analysis of the basic toxicokinetics will be a qualitative assessment based on phys/chem properties and available biological information on ethyl caproate and structurally-related analogs according to the guidance provided in the ECHA guidelines (ECHA, 2014). 

Since the target substance is a small aliphatic ester, it is subject to hydrolysis to the corresponding alcohol and carboxylic acid. For ethyl caproate these areethyl alcohol (ethanol, CAS 64-17-5) and hexanoic acid (CAS 142-62-1).). The hydrolysis facilitates absorption, metabolism and excretion by generating smaller size molecules from the parent ester. This hydrolysis is facilitated by esterases which are present in nearly every mammalian tissue (WHO, 1997; Jewellet al., 2010; Oslonet al.,1993). 

Hydrolysis studies with artificial pancreatic juice demonstrated ethyl caproate is rapidly hydrolyzed with a half-life (t_½) of 3 minutes (Longlandet al.,1977). Thus, hydrolysis of ethyl caproate is predicted to result in a short residence time of intact ester in the gastrointestinal (GI) tract after ingestion. Because of the ubiquitous nature of esterases in the body, similar rates of hydrolysis would be expected in other tissue compartments as well. Thus, by any route of administration, esterase-mediated hydrolysis would be likely.

Table1Physical/chemical properties of ethyl caproate and hydrolysis products relevant to toxicokinetics

Physicochemical endpoints	Ethyl caproate Target	Ethanol* Hydrolysis Product	Hexanoic acid* Hydrolysis Product
CAS	123-66-0	64-17-5	142-62-1
EC	204-640-3	200-578-6	271-676-4
Molecular formula	C8H16O2	C2H6O	C6H12O2
Molecular weight	144.2	46.1	116.2
Physical state	liquid	liquid	liquid
Melting point (°C)	-68.3	-114	-3
Boiling point (°C)	167.3	78.4	206
Density (g/cm^3)	0.871 @ 20 °C	0.79 @ 20 °C	0.93 @ 20 °C
Vapor pressure (hPa)	3 @ 20 °C	59.5 @ 20 °C	0.27 @ 20 °C
Water solubility (mg/L)	508 @ 20° C pH 4.5	miscible	1100 @ 20° C
Partition coefficient log Kow	2.96	-0.18	1.92

*Information obtained from Pub Chem -https://pubchem.ncbi.nlm.nih.gov

Toxicokinetics

The toxicokinetics of absorption, distribution, metabolism and excretion (ADME) of ethyl caproate have not been evaluatedinvivo. As a result, this analysis of the basic toxicokinetics will be a qualitative assessment based on phys/chem properties and available biological information on ethyl caproate and structurally-related analogs according to the guidance provided in the ECHA guidelines (ECHA, 2014). 

Since the target substance is a small aliphatic ester, it is subject to hydrolysis to the corresponding alcohol and carboxylic acid. For ethyl caproate these areethyl alcohol (ethanol, CAS 64-17-5) and hexanoic acid (CAS 142-62-1).). The hydrolysis facilitates absorption, metabolism and excretion by generating smaller size molecules from the parent ester. This hydrolysis is facilitated by esterases which are present in nearly every mammalian tissue (WHO, 1997; Jewellet al., 2010; Oslonet al.,1993). 

Hydrolysis studies with artificial pancreatic juice demonstrated ethyl caproate is rapidly hydrolyzed with a half-life (t_½) of 3 minutes (Longlandet al.,1977). Thus, hydrolysis of ethyl caproate is predicted to result in a short residence time of intact ester in the gastrointestinal (GI) tract after ingestion. Because of the ubiquitous nature of esterases in the body, similar rates of hydrolysis would be expected in other tissue compartments as well. Thus, by any route of administration, esterase-mediated hydrolysis would be likely.

Mode of Action

No mode of action for adverse effects has been developed for ethyl caproate. Studies available to date have not identified a target organ and no significant adverse effects have been noted by which a mode of action can be derived. In addition, there are no structural alerts with ethyl caproate or hexanoic acid products which could be used to hypothesize a mode of action. The metabolism of ethanol results in the formation reactive oxygen species, which can, in excess result in cellular oxidative stress if these reactive molecules are present at levels which cannot be dealt with by anti-oxidant species.  This process is considered to be a key event in the development of alcohol–induced liver disease. The exposure to ethanol resulting from the use of ethyl caproate is highly unlikely to result in the levels these molecules would need to be present to cause cellular injury.

Toxicodynamics

The biological effects of ethyl caproate are limited. As a result, no toxicodynamic effects have been described.

Applicant's summary and conclusion

Conclusions:

Conclusions
1. Ethyl caproate can be assumed to be absorbable by the oral, dermal, and inhalation routes of exposure.
2. Ethyl caproate is rapidly and completely hydrolyzed to form ethanol and hexanoic acid.
3. Both ethanol and hexanoic acid are readily incorporated into intermediary metabolism.
4. There is no evidence of the formation of reactive or toxic metabolites from hexanoic acid and given the low potential exposure, ethanol intoxication is unlikely.
5. Ethyl caproate is unlikely to bioaccumulate because of its extensive metabolism and rapid excretion.

Executive summary:

Ethyl caproate is a small aliphatic ester with a molecular weight of 144.2 g/mole. It has limited water solubility (508 mg/L at 20°C, pH 4.9) with a partition coefficient (Log P_ow) of 2.96 and a vapor pressure of 3 hPa at 20°C. With these physical/chemical (phys/chem) properties, oral, dermal and inhalation exposures are all potential routes of exposure. 

The metabolism and excretion of ethyl caproate has been evaluated by the WHO Expert Committee on Food Additives in a review of food additives and contaminants (WHO 1997). Ethyl caproate is readily hydrolyzed to the corresponding ethyl alcohol (ethanol, CAS 64-17-5) and aliphatic carboxylic acid, hexanoic acid (CAS 142-62-1). Ethanol is oxidized by alcohol and aldehyde dehydrogenases to eventually form acetyl CoA which joins the natural carbon pool to be converted into CO₂, fatty acids, ketone bodies and cholesterol (Cederbaum, 2012). Hexanoic acid may be conjugated with glucuronic acid and excreted in the urine, excreted directly due to its size and water solubility or be incorporated into intermediary metabolism and utilized nutritionally via fatty acid metabolic pathways.

Ethyl caproate has limited chemical and biological reactivity and no defined mode of action for adverse effects. The oral LD₅₀in rats is > 5000 mg/kg oral and > 2000 mg/kg dermal. It is non-irritating to the eye, is not a dermal sensitizer, not genotoxic, has no structural alerts for protein or DNA binding and no alerts for cancer. Ethyl caproate is classified as a skin irritant (H315) based on anin vitroEpiderm™ study. As a result, there is no basis for identifying analogs based on common systemic modes of action. Therefore, selection of analogs for read-across to predict toxicokinetics is based solely on structural similarity.