3-methylbutyl butyrate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Type of information:

other: Evaluation of toxicokinietcs based on literature data

Adequacy of study:

other information

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
Isoamyl butyrate possesses the phys/chem properties that favour 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 (ECHA, 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 isoamyl butyrate is 158.24 g/mole which would favour its absorption from the GI tract. In addition, the test item is moderately water soluble (184.7 mg/L at 20°C, pH 5.8)) and has an octanol-water partition coefficient (log Pow) of 3.99. This combination of aqueous and lipid solubility also generally favours absorption by the oral route. However, the test item is readily hydrolysed in the GI tract which would effectively reduce the availability of the test item for absorption. The hydrolysis of isoamyl butyrate within the GI tract has been investigated and half-lives (t1/2) between 0.1 seconds (intestinal homogenate) and 11 hours (artificial gastric juice) were reported. In the presence of artificial pancreatic juice, isoamyl butyrate was reported to have been 100 % hydrolysed after 2 hours (Longland et al., 1977). Based on these data it is reasonable to conclude most of the ingested test item will be hydrolysed before it could be absorbed and reach systemic circulation.

Finally, any intact test item that is absorbed would be almost immediately hydrolysed by esterases in blood. Rapid hydrolysis of isoamyl-3-(2’-furyl) propionate (CAS 7779-67-1), allyl phenylacetate (CAS 1797-74-6) and 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 were monitored. No intact ester was detected in the blood.
Based on this, the biological effects from ingestion of isoamyl butyrate would be predicted to result from exposure to the isoamyl alcohol and the butyric acid formed from the hydrolysis of the test item. In an acute oral toxicity test, a single dose of isoamyl butyrate (5000 mg/kg bw) was administered to a group of 10 rats. Clinical observations including slight lethargy and diarrhea were observed. These effects were considered not to be of toxicological relevance but they do indicate systemic exposure following oral administration of the test item.
In acute oral toxicity tests where the hydrolysis products are administered, similar clinical observations to the parent ester have been observed. Isoamyl alcohol was administered to the rat (3/sex/dose) by the oral route for 14 days at 0, 15, 60, 250 or 1000 mg/kg bw/day. At 1000 mg/kg bw/day slight sedation in behaviour was observed for up to 30 minutes after dosing (ECHA, 2017a). In a 7 day repeat dose dietary study in the rat, butyric acid administered at a concentration of 4 % in the diet (no further data reported), adverse changes to the GI tract were observed (ECHA, 2017b).
Based on the available information, it is reasonable to assume that 100 % of isoamyl butyrate, or its hydrolysis products, will gain systemic circulation by the oral route of absorption. 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, the isoamyl butyrate 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. The molecular weight of isoamyl butyrate is 158.24 g/mole which satisfies this guideline. In addition, both water solubility (184.7 mg/L at 20°C, pH 5.8) and log Pow (3.99) 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 (Kp) 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 of the test item is 3.99 and the molecular weight is 158.24.
The resulting dermal permeability coefficient, (Log Kp) is -0.85 cm/hr (Kp = 0.14 cm/hr).

This permeability coefficient can be used to estimate the amount of isoamyl butyrate absorbed when applied to the skin. The following equation utilizes the 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 184.7 mg/L at 20°C (pH 5.8). 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 isoamyl butyrate the ADR is 0.003 mg/cm2/hr or 3 ug/cm2/hr. Thus, dermal absorption of the test item would be expected to result in systemic exposure. However, once the test item 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.

In an acute dermal toxicity test, a single dose of isoamyl butyrate (5000 mg/kg bw) was administered to a group of 10 rabbits. Clinical observations were limited to two incidents of diarrhea. Since there was no lethality from the test item 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.
The chemical properties for isoamyl butyrate are well within the ECHA guidelines to favour dermal absorption. However, 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
Isoamyl butyrate is a liquid at room temperature with limited volatility. The vapour pressure of the test item is 1.9 hPa at 25° C which limits the likelihood of significant systemic exposure by inhalation of vapours. However, if the test item were aerosolized, absorption across the respiratory epithelium would likely be 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 butyrate 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. 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 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 butyric acid. Both materials are expected to freely distribute systemically and enter intermediary metabolism in all tissues. Therefore, the distribution of isoamyl alcohol and butyric acid would be proportional to organ blood flow. After administration by the oral route, the hydrolysis products would enter hepatic portal circulation, resulting in primary distribution to the liver (Annison and Leng, 1963). 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 isoamyl alcohol and butyric acid. For isoamyl alcohol, a glucuronide conjugate 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 butyric acid is consumed by fatty-acid metabolism pathways via ß-oxidation and is utilized nutritionally (Pouteau et al., 2003).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The test item is expected to be rapidly and complexly hydrolysed to isoamyl alcohol and butyric acid 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 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 favoured, but with all these potential pathways, the biotransformation occurs without the formation of reactive intermediates, which could lead to adverse effects.

Butyric acid (CAS: 107-92-6): This straight chain carboxylic acid is used endogenously as part of the citric acid cycle (Leschelle et al., 2000). This compound may also be used as 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.

Any other information on results incl. tables

This analysis of the basic toxicokinetics is a qualitative assessment based on physicochemical properties and available biological information on isoamyl butyrate and structurally-related analogs according to the guidance provided in the ECHA guidelines (ECHA, 2014).

 

Since the target substance is a small branched aliphatic ester, it is subject to hydrolysis to the corresponding alcohol and carboxylic acid. For isoamyl butyrate these are isoamyl -alcohol (CAS 123-51-3) and butyrate (CAS 107-92-6), respectively. 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; Oslon et al.,1993). Hydrolysis studies with artificial pancreatic juice demonstrate that isoamyl butyrate is rapidly hydrolysed with a half-life (t½) of 11 minutes (Longland et al., 1977). Thus, hydrolysis of isoamyl butyrate 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.

 

 

Table 1: Physical/chemical properties of isoamyl butyrate and hydrolysis products relevant to toxicokinetics: 

Physicochemical endpoints	Isoamyl butyrate Target	Isoamyl alcohol* Hydrolysis product	Butyric acid* Hydrolysis product
CAS	106-27-4	123-51-3	107-92-6
EC	203-380-8	204-633-5	203-535-3
Molecular formula	C9H18O2	C5H12O	C4H802
Molecular weight (g/mol)	158.24	88.15	88.12
Physical state	liquid	liquid	liquid
Melting point^oC	< -100°C	-117.2	-5.7
Boiling point^oC	180.6°C	132.2	163.7
Density (g/cm^3)	0.86 (relative density)	0.81@ 15^oC	0.96 @ 20^oC
Vapour Pressure (hPa)	1.9 @ 25.0°C	3.16 @ @ 25^oC	5.7 @ 25^oC
Water solubility (mg/L)	184.7mg/l @ 20.0°C	26700 @ 25^oC	60000 @ 25^oC
Partition coefficient log Pow	3.99@22.4°C	1.16	0.79

*Data source: ChemID: A toxnet database. Available at chem.nlm.nih.gov

 

 

Applicant's summary and conclusion

Conclusions:

Conclusions
1. The test item 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. The test item is rapidly and completely hydrolyzed to form to isoamyl alcohol and butyric acid.
3. Both to isoamyl alcohol and butyric acid are readily incorporated into intermediary metabolism.
4. There is no evidence of the formation of reactive or toxic metabolites from either to isoamyl alcohol and butyric acid.
5. The test item is unlikely to bioaccumulate because of its extensive metabolism and rapid excretion.

Executive summary:

Isoamyl butyrate is a small aliphatic ester with a molecular weight of 158.24 g/mole. It has a limited water solubility (184.7 mg/L at 20°C, pH 5.8) with an estimated partition coefficient (Log Pow) of 3.99 (pH 7.0) and a vapour pressure of 1.9 hPa at 25^°C. 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). Isoamyl butyrate is readily hydrolysed to the corresponding branched alcohol, isoamyl alcohol (CAS 123-51-3) and aliphatic carboxylic acid, butyric acid (CAS: 107-92-6): 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 butyric acid 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,does not have structural alerts for sensitization and structurally related analogs are not dermal sensitizers, 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.