Registration Dossier

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

Data source

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Oral
Ethyl isovalerate (EIV) 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 EIV 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 EIV is 130.2 g/mole which would favor its absorption from the GI tract. In addition, EIV is water soluble (1.8 g/L) and has an octanol-water partition coefficient (log Pow) of 2.5. This combination of aqueous and lipid solubility also generally favors absorption by the oral route.
However, EIV is readily hydrolyzed in the GI tract which would effectively reduce the availability of EIV for absorption. Given the relatively short t1/2 of EIV in the GI tract (2 min) and the GI residence time (24-72 hours in humans) it is reasonable to conclude most of the ingested EIV will be hydrolyzed before it could be absorbed and reach systemic circulation. Assuming there is an excess of esterases in the GI tract, the hydrolysis of EIV would obey linear first-order kinetics resulting in 98% of the ingested EIV undergoing hydrolysis within 60 minutes.
Finally, any intact EIV 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.
The biological effects from ingestion of EIV would be predicted to result from exposure to the ethyl alcohol and the isovaleric acid formed from the hydrolysis of EIV.
There are no bioanalytical or clinical findings from acute or repeat-dose studies available to substantiate the projections that EIV would be absorbed after oral administration. All of the studies conducted to date on EIV have not resulted in effects which can be attributed to oral administration of EIV.
Based on the available information, it is reasonable to assume that 100% of the ethyl isovalerate, or its hydrolysis products, will gain systemic circulation by the oral route of administration.
Although the test item can be expected to be well absorbed by the oral route, in the absence of experimental absorption data, default values are used in the derivation of DNELs according to REACH guidance R.8 page 19. For oral absorption this is 50%.

Dermal
Based on the phys/chem properties, EIV is likely to be absorbed after dermal application. According to the ECHA Guidelines (ECHA 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 is one of the most widely applied (US EPA 2007). 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 2.5 and the molecular weight is 130.2. The resulting dermal permeability coefficient, (Log Kp) is -1.74 cm/hr (Kp = 0.0184 cm/hr).

This permeability coefficient can be used to estimate the amount of EIV 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

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 EIV in water is 1.8 g/L (1800 mg/L) at 20° C. For the surface area one cm2 is used to generate a flux vale that can be applied across a variety of applications with different surface area values.

For EIV the ADR is 32.8 mg/cm2/hr. Thus, dermal absorption of EIV would be expected. However, once the parent EIV crosses the stratum cornea, the esterases present in epidermal cells (Jewell et al 2007) would be expected to hydrolyze the EIV to its corresponding alcohol and carboxylic acid.

Since there was no lethality from EIV in the dermal acute toxicity assays 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 absorbed by the dermal route, in the absence of experimental absorption data, default values are used in the derivation of DNELs according to REACH guidance R.8 page 19. For dermal absorption this is 50%.

Inhalation
Inhalation EIV is a liquid at room temperature with limited volatility. The vapor pressure of EIV is 10.4 hPa at 20° C which limits the likelihood of significant systemic exposure by inhalation of vapors. However, if EIV 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 ethyl alcohol and isovalerate 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 substantial the prediction that EIV is absorbed after inhalation.

Based on the available information, it is reasonable to assume that 100% of the ethyl isovalerate, or its hydrolysis products, will gain systemic circulation after inhalation.

Although the test item can be expected to be absorbed by the inhalation route, in the absence of experimental absorption data, default values are used in the derivation of DNELs according to REACH guidance R.8 page 19. For inhalation absorption this is 100%.



Details on distribution in tissues:
The distribution of EIV has not been characterized. The systemic effects noted after oral dosing are minimal and not specific enough to determine a distribution of absorbed EIV. However, as noted, by all routes of exposure, significant hydrolysis of EIV is expected forming ethyl alcohol and isovalerate. Both materials are expected to freely distribute systemically and enter intermediary metabolism in all tissues. Therefore, the distribution of ethyl alcohol and isovalerate 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 EIV enter into intermediary metabolism it is unlikely that either would bioaccumulate.
Details on excretion:
The excretion kinetics of EIV are dependent on the hydrolysis of the ester and the metabolic fate of the ethyl alcohol and isovalerate. For ethanol, the majority of the dose is expected to be metabolized and incorporated into endogenous metabolic pathways (Cederbaum, 2012). The isovalerate is consumed by fatty-acid metabolism pathways via ß-oxidation and is utilized nutritionally. Only the branched remnant is eliminated in the urine (Belsito, 2010).

Metabolite characterisation studies

Details on metabolites:
EIV is expected to be rapidly and complexly hydrolyzed to ethyl alcohol and isovalerate either before, or immediately after absorption by all routes of exposure. Both of these hydrolysis products become substrates for intermediary metabolism.

Ethyl alcohol (64-17-5) This molecule can be directly conjugated to a glucuronide or sulfate and excreted in the urine. It can also be converted to the aldehyde (acetaldehyde) by alcohol dehydrogenase (Cederbaum, 2012). The acetaldehyde is enzymatically converted to Acetyl-Co A and enters the endogenous 2-carbon pool of the citric-acid cycle.
Isovalerate (503-74-2) This branched carboxylic acid is a substrate for endogenous fatty-acid metabolism that occurs via ¿-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. In branched fatty acids, such as isovalerate, the process continues until the it reaches the branched carbon and the process stops. The resulting branched CoA conjugate is then excreted in the urine.

Any other information on results incl. tables

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

Physicochemical endpoints

Ethyl isovalerate

Target

Ethanol*

Hydrolysis Product

Isovaleric acid *

Hydrolysis Product

CAS

108-64-5

64-17-5

503-74-2

EC

203-602-3

200-578-6

207-975-3

Molecular formula

C7-H14-O2

C2H6O

C5-H10-O2

Molecular Weight

130.2

46.1

102.1

Physical state

liquid

liquid

liquid

Melting point (°C)

NA

-114

-29.3

Boiling point (°C)

135.3

78.4

176.5

Density (g/cm3)

0.865 @ 20 °C

0.79 @ 20 °C

0.93 @ 20 °C

Vapor pressure (hPa)

10.4 @ 20 °C

59.5 @ 20 °C

0.6 @ 20 °C

Water solubility (g/L)

1.8 @ 20° C pH 6.5

miscible

40.7 @ 20° C

Partition coefficient log Kow

2.5

-0.18

1.16

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

Toxicokinetics

The toxicokinetics of absorption, distribution, metabolism and excretion (ADME) of EIV have not been evaluatedinvivo. As a result, this analysis of the toxicokinetics will be a qualitative assessment based on phys/chem properties and available biological information on EIV 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 EIV the corresponding alcohol is ethyl alcohol (CAS64-17-5) and the corresponding carboxylic acid is isovalerate (CAS503-74-2). 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, Jewell et al 2007, Olsonet al1993). 

Hydrolysis studies with liver homogenate demonstrated EIV is hydrolyzed with a half-life (t1/2) of 24 seconds (Longland,et al 1977). In the same report, the half-life generated by intestinal homogenate was 2 minutes. Thus, hydrolysis of EIV is predicted to result in a short residence time of EIV parent in the GI tract after ingestion. Because of the ubiquitous nature of esterases in the body, 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 have been developed for EIV. 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 EIV or its two hydrolysis products which could be used to hypothesize a mode of action. 

The metabolism of ethanol results in the formation reactive electrophiles such as acetaldehyde or reactive oxygen species (ROS), which can, in excess, result in adverse effects resulting from protein binding or oxidative stress. 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 EIV is unlikely to result in the levels these reactive species at concentrations necessary to cause cellular injury.

Toxicodynamics

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

Applicant's summary and conclusion

Conclusions:
1. EIV can be assumed to be absorbed by the oral, dermal, and inhalation routes of exposure. However in the absence of experimental data, default absorption values are used in the derivation of DNELs, according to REACH guidance R.8 page 19.
2. EIV is rapidly and completely hydrolyzed to form ethyl alcohol and isovalerate.
3. Both ethyl alcohol and isovalerate are readily incorporated into intermediary metabolism.
4. There is no evidence of the formation of significant levels of reactive or toxic metabolites from either ethyl alcohol or isovalerate.
5. EIV is unlikely to bioaccumulate because of its extensive metabolism and rapid excretion.
Executive summary:

Ethyl isovalerate (EIV) is a small aliphatic ester with a molecular weight of 130.2 g/mole. It is soluble in water at up to 1.8 g/L at 20¿C with a partition coefficient (Log Pow) of 2.5 at 24¿C and a vapor pressure of 10.4 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 EIV has been evaluated by the WHO Expert Committee on Food Additives in a review of food additives and contaminants. EIV is readily hydrolyzed to the corresponding alcohol, ethyl alcohol (CAS 64-17-5) and branched carboxylic acid, isovaleric acid (CAS 503-74-2). Once formed, both of these substances can be glucuronidated and excreted in the urine or, because of their small size and water solubility, excreted directly in the urine. However, the remaining ethyl alcohol and isovalerate can also be incorporated into intermediary metabolism and utilized nutritionally via the endogenous 2-carbon pool andviaamino-acid and fatty-acid metabolic pathways.

EIV has limited chemical and biological reactivity and no defined mode of action for adverse effects. The oral LD50in rats is > 5,000 mg/kg oral and > 2000 mg/kg dermal. It is non-irritating to skin and eye, does not have structural alerts for sensitization and structurally related analogs are not dermal sensitizers. It is not genotoxic, has no structural alerts for protein or DNA binding and no alerts for cancer. As a result, there is no basis for identifying analogs based on common modes of action. Therefore, selection of analogs for read-across to predict toxicokinetics is based solely on structural similarity.