3-methylbutyl isovalerate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Type of information:

other: Evaluation of toxicokinietcs based on literature data

Adequacy of study:

key study

Study period:

NA

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

ORAL
The test item 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 the test item 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 IVIA is 172.3 g/mole which would favor its absorption from the GI tract. In addition, the test item is moderately water soluble (48.1 mg/L) and has an octanol-water partition coefficient (log Pow) of 3.8. This combination of aqueous and lipid solubility also generally favors absorption by the oral route.
However, the test item is readily hydrolyzed in the GI tract which would effectively reduce the availability of the test item for absorption. Given the relatively short t1/2 of the test item in the GI tract (10 min) and the GI residence time (24-72 hours in humans) it is reasonable to conclude most of the ingested test item 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 IVIA would obey linear first-order kinetics resulting in 98% of the ingested IVIA undergoing hydrolysis within 60 minutes.
Finally, any intact test item 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 the test item would be predicted to result from exposure to the isoamyl alcohol and the isovaleric acid formed from the hydrolysis of the test item.
In a two-week repeat-dose study in rats IVIA was administered by oral gavage at up to 1000 mg/kg bw/d. There was an increase in salivation and a decrease in body weight gain in the high-dose group without a corresponding decrease if food consumption (KIT 2017). This combination of findings is an indicator that systemic exposure occurred after oral dosing. In a 90-day dietary study at doses up to 219 mg/kg bw/d there were no adverse effects noted and only a slight increase in blood glucose levels which were biologically insignificant (FEMA 1997). Finally, in a 50-day pre-natal developmental study in rats, there were no adverse effects noted after oral dosing at up to 800 mg/Kg bw but, as in the two week study, there was excess salivation noted in the 800 mg/kg bw group (KIT 2017). There was no corresponding decrease in body weight gain in this study.
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, the test item 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 3.8 and the molecular weight is 172.3. The resulting dermal permeability coefficient, (Log Kp) is -1.073 cm/hr (Kp = 0.084 cm/hr).
This permeability coefficient can be used to estimate the amount of IVIA 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 the test item in water is 48.1 mg/L at 20° C and skin pH (6.5). 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 IVIA the ADR is 0.004 mg/cm2/hr or 4 ug/cm2/hr. Thus, dermal absorption of IVIA would be expected to result in systemic exposure. However, once the parent IVIA crosses the stratum cornea, the esterases present in epidermal cells (Jewell et al 2007) would be expected to hydrolyze the test item to its corresponding alcohol and carboxylic acid.
Since there was no lethality from the test item 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 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:
IVIA is a liquid at room temperature with limited volatility. The vapor pressure of the test item is 1.3 hPa at 20° C which limits the likelihood of significant systemic exposure by inhalation of vapors. However, if the test item 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 isoamyl 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 the test substance available.
In the absence of experimental data a default inhalation absorption value of 100% is used in derivation of DNELs

Details on distribution in tissues:

The distribution of the test item has not been characterized. The systemic effects noted after oral dosing are minimal and not specific enough to determine a distribution of absorbed test item. However, as noted, by all routes of exposure, significant hydrolysis of the test item is expected forming isoamyl alcohol and isovalerate. Both materials are expected to freely distribute systemically and enter intermediary metabolism in all tissues. Therefore, the distribution of isoamyl 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 the test item enter into intermediary metabolism it is unlikely that either would bioaccumulate.

Details on excretion:

The excretion kinetics of the test item are dependent on the hydrolysis of the ester and the metabolic fate of the isoamyl alcohol and isovalerate. For isoamyl alcohol only the portion of the molecule that is conjugated to the glucuronide is excreted in the urine. This appears to be a relatively minor pathway as only 9% of isoamyl alcohol administered to rats is recovered as the glucuronide conjugate in the urine (Belsito et al 2010). The remaining isoamyl alcohol is incorporated into the leucine catabolism pathway and utilized nutritionally in the citric acid cycle. 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.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The test item is expected to be rapidly and complexly hydrolyzed to isoamyl alcohol and isovalerate either before, or immediately after absorption by all routes of exposure. Both of these hydrolysis products become substrates for intermediary metabolism.

Isoamyl alcohol (CAS 123-51-3): This molecule is enzymatically can be directly conjugated to a glucuronide or sulfate and excreted in the urine. It can also be converted to the aldehyde by alcohol dehydrogenase (Belsito et al, 2010). The formed aldehyde can be further oxidized to the corresponding acid or conjugated by UDP-glucuronosyltransferase. In both cases, the biotransformed products would be excreted in the urine. The remaining aldehyde would also be a substrate for intermediary metabolism in the endogenous metabolism of leucine (Rosenthal et al, 2007). There is no information available to know which of the various pathways predominate or are kinetically favored but with all these potential pathways the biotransformation occurs without the formation of reactive intermediates which could lead to adverse effects.

Isovalerate (CAS 503-74-2): This branched carboxylic acid is a substrate for endogenous fatty-acid metabolism that occurs via beta-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 isoamyl isovalerate and hydrolysis products relevant to toxicokinetics

Physicochemical endpoints	Isoamyl isovalerate Target	Isoamyl alcohol* Hydrolysis Product	Isovaleric acid* Hydrolysis Product
CAS	659-70-1	123-51-3	503-74-2
EC	211-536-1	204-633-5	207-975-3
Molecular formula	C10-H20-O2	C5H12O	C5-H10-O2
Molecular Weight	172.3	88.2	102.1
Physical state	liquid	liquid	liquid
Melting point (°C)	NA	-117	-29.3
Boiling point (°C)	192.2	132.3	176.5
Density (g/cm^3)	0.855 @ 20 °C	0.81 @ 20 °C	0.93 @ 20 °C
Vapor pressure (hPa)	1.3 @ 20 °C	37.3 @ 20 °C	0.6 @ 20 °C
Water solubility (mg/L)	48.1 @ 20° C pH 6.5	26700 @ 20° C	40700 @ 20° C
Partition coefficient log Kow	3.8	1.16	1.16

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

Toxicokinetics

The toxicokinetics of absorption, distribution, metabolism and excretion (ADME) of isoamyl isovalerate have not been evaluated in vivo. As a result, this analysis of the toxicokinetics will be a qualitative assessment based on phys/chem properties and available biological information on isoamyl isovalerate 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 isoamyl isovalerate the corresponding alcohol is isoamyl alcohol (CAS123-51-3) 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 2010, Oslonet al1993).  Hydrolysis studies with artificial pancreatic juice demonstrated isoamyl isovalerate is rapidly hydrolyzed with a half-life (t_1/2) of 10 minutes (Longland,et al 1977). Isoamyl butyrate, a structurally similar analog, had a similar t_1/2of 11 min is the same preparation. Thus, hydrolysis of isoamyl isovalerate is predicted to result in a short residence time in the 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 isoamyl isovalerate. 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 isoamyl isovalerate or its two hydrolysis products which could be used to hypothesize a mode of action.

Toxicodynamics

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

Applicant's summary and conclusion

Conclusions:

1. The test item can be assumed to be absorbed by the oral, dermal, and inhalation routes of exposure, however default absorption values are used in the derivation of DNELs, according to REACH guidance R.8 page 19
2. The test item is rapidly and completely hydrolyzed to form isoamyl alcohol and isovalerate.
3. Both isoamyl alcohol and isovalerate are readily incorporated into intermediary metabolism.
4. There is no evidence of the formation of reactive or toxic metabolites from either isoamyl alcohol or isovalerate.
5. The test item is unlikely to bioaccumulate because of its extensive metabolism and rapid excretion.

Executive summary:

Isoamyl isovalerate is a small aliphatic ester with a molecular weight of 172.3 g/mole. It has limited water solubility (48 mg/L) with a partition coefficient (Log P_ow) of 3.8 at pH 7.4 and a vapor pressure of 1.3 hPa at 20oC. With these physical/chemical (phys/chem) properties, oral, dermal and inhalation exposures are all potential routes of exposure.  For the derivation of DNELs default absorption values are used according to REACH guidance R.8 p.19.

The metabolism and excretion of the test item has been evaluated by the WHO Expert Committee on Food Additives in a review of food additives and contaminants (WHO 1997). IAIV is readily hydrolyzed to the corresponding branched alcohol, isoamyl alcohol (CAS 123-51-3) 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 isoamyl alcohol and isovalerate can also be incorporated into intermediary metabolism and utilized nutritionally via amino-acid and fatty-acid metabolic pathways.

The test item

has limited chemical and biological reactivity and no defined mode of action for adverse effects. The oral LD₅₀ in rats is > 5,000 mg/kg oral and > 2000 mg/kg dermal. It is non-irritating to skin and eye, is not a dermal sensitizer, 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.