Phenethyl isobutyrate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics, other

Remarks:

Expert statement

Type of information:

other: Expert statement

Adequacy of study:

key study

Study period:

2016-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Expert statement, no study available

Principles of method if other than guideline:

Expert statement

GLP compliance:

no

Details on test animals or test system and environmental conditions:

not applicable

Details on exposure:

not applicable

Duration and frequency of treatment / exposure:

not applicable

Remarks:

Doses not applicable

No. of animals per sex per dose / concentration:

not applicable

Positive control reference chemical:

not applicable

Details on study design:

not applicable

Details on dosing and sampling:

not applicable

Statistics:

not applicable

Details on absorption:

Generally, oral absorption is favoured for molecular weights below 500 g/mol. Moderate water solubility of 160 mg/L enables the substance to partly dissolve in the gastrointestinal fluids and thus it may be considered that direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across membranes with the bulk passage of water takes place. However, the moderate log Pow value of 3.5, which is favourable for passive diffusion, would be the more important property here regarding absorption. Therefore, it can be concluded that Phenylethyl Isobutyrate likely becomes bioavailable following the oral route, as indicated by its physic chemical properties. This assumption is confirmed by the results of the acute oral toxicity study as well as the combined repeated dose toxicity study (OECD 422). In the acute toxicity study, considerable clinical signs, as well as mortalities were observed following administration (leading to LD50 values of >5000 mg/kg bw).

Due to the low vapour pressure of 45 Pa of Phenylethyl Isobutyrate it is unlikely that the substance will be available as a vapour. However, it this case, absorption via inhalation route would be possible due to the moderate log Pow value, enabling uptake directly across the respiratory tract epithelium by passive diffusion.

Dermal absorption can also take place, favoured by the log Pow value, and also by the small molecular weight of the substance. Indeed, clinical effects and mortality at the limit dose of 5000 mg/kg bw were reported (please refer to IUCLID section 7.2.3).

Details on distribution in tissues:

As mentioned above, the physicochemical properties of Phenylethyl Isobutyrate make systemic bioavailability very likely following oral, inhalative and dermal uptake.
After being absorbed into the body, Phenylethyl Isobutyrate is likely distributed into cells and intracellular concentrations may be higher than extracellular concentrations due to its slightly lipophilic properties (log Pow 3.5). This applies especially for fatty tissues.
Phenylethyl Isobutyrate is unlikely to have a bio-accumulative potential, because it is not highly lipophilic (log Pow is not greater than 4) and there are no other physic-chemical properties indicating bio-accumulating properties. As a combined repeated dose toxicity study (OECD 422) in rats did not reveal any signs of target organ toxicity or other indications for an accumulation in any organ or tissue, there is also no evidence for an accumulative property of this compound. Furthermore, it is stated in literature that the breakdown products are readily metabolized and excreted.

Details on excretion:

Phenylethyl Isobutyrate will not be excreted in its non-hydrolysed form. One degradation product, phenylethyl alcohol, is excreted in the urine as conjugate with glycine or glucuronic acid (JECFA, 2003). The other hydrolysis product, isobutyric acid, is rapidly metabolised, integrated in endogenous pathways and thus in main parts excreted as CO2 in the breath (DiVincenzo & Hamilton, 1979). The assumption of rapid excretion is supported by findings of the acute oral toxicity study as well as the combined repeated dose study (OECD 422). In both studies surviving animals recovered rapidly when treatment was determined.

Details on metabolites:

Based on its structure, Phenylethyl Isobutyrate may be considered to possibly undergo hydrolysis in contact with aqueous fluids into the corresponding carboxylic acid (isobutyric acid) and phenylethyl alcohol. This process is also highly likely catalyzed by carboxylesterases, (Mutschler, 2001). Hydroxyl group and carboxylic acid are typical functional groups of substrates for phase II enzymes such as UGTs (UDP-glucuronosyltransferase) and GSTs (glutathione-S-transferases) (Marquat & Schäfer, 2004). Conjugated phenylethyl alcohol with glucuronic acid as well as with glycine was already demonstrated in the urine of rabbits (JECFA, 2003). Isobutyric acid, however, is conjugated with Co-enzyme-A and, following β-oxidation, integration into endogenous citric acid cycle is considered (BG Chemie, 1997). Summarizing, the substance and its hydrolytic cleavage products are considered to be readily metabolized and, with this, detoxified. This consideration is supported by findings of in vitro genotoxicity studies applying a metabolizing system (mammalian S9 mix). In both the bacterial reverse mutation assay and the in vitro micronucleus assay the substance showed no cytotoxicity when incubated together with S9. However, substantial cytotoxicity was observed when the substance was applied without metabolizing system in both assays. This is further supported by an HPRT assay, where cytotoxicity was observed irrespective of S9 application or not.

Conclusions:

Bioaccumulation of the substance is not to be excpected after continuous exposure based on expert statement.

Executive summary:

Based on physicochemical characteristics, particularly molecular weight and octanol-water partition coefficient, absorption by the dermal, oral and inhalation route is expected. This assumption is further supported by the results of the oral and dermal acute toxicity studies, revealing effects at very high doses (5000 mg/kg bw), as well as the combined repeated dose study (OECD 422). Bioaccumulation of Phenylethyl Isobutyrate or its breakdown products phenylethyl alcohol or isobutyric acid will not occur. Enzymatic hydrolysis of Phenylethyl Isobutyrate is supposed to be complete. Phenylethyl alcohol is conjugated with glucuronic acid or glutathione and excreted via urine. Isobutyric acid is metabolised, incorporated in endogenous pathways and excreted as CO2 in the breath.

Description of key information

Based on physicochemical characteristics, particularly molecular weight and octanol-water partition coefficient, absorption by the dermal, oral and inhalation route is expected. This assumption is further supported by the results of the oral and dermal acute toxicity studies, revealing effects at very high doses (5000 mg/kg bw), as well as the combined repeated dose study (OECD 422). Bioaccumulation of the test item or its breakdown products will not occur. Enzymatic hydrolysis of the test item is supposed to be complete. The break down products are either conjugated with glucuronic acid or glutathione and excreted via urine or metabolised, incorporated in endogenous pathways and excreted as CO2 in the breath.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Toxicological profile of Phenylethyl Isobutyrate

Acute oral toxicity study conducted in rats revealed an LD50 value of >5000 mg/kg bw. No data are available on acute toxicity after inhalation. In an acute dermal toxicity study conducted with Phenylethyl Isobutyrate in rats the LD50 value was also found to be greater than 5000 mg/kg bw.

 

Phenylethyl Isobutyrate was also concluded to be not irritating to skin or eyes based on in vitro and in vivo test results with the substance. In a weight of evidence approach taking two in vitro studies into account, no skin sensitising properties were revealed.

 

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422) in rats, treatment related adverse effects and signs of toxicity towards reproduction were found at the highest tested dose of 1000 mg/kg bw/day only. Rats were dosed daily by oral gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/d, respectively. Treatment period covered 14 days pre-mating and up to 14 days during the mating period, followed by a 14 days of recovery, and during gestation on pregnant females from gestation day 0 to day 4 post-partum. Effects of general toxicity were observed at the high dose only and included clinical signs of salivation in both sexes and reduced body weight gain in males, significant changes in blood and clinical chemistry parameters in both sexes and males only, microscopic changes in the liver, thymus and testes. Treatment with test item at the highest dose also revealed effects on reproduction, such as reduced gestation and higher mortality indices, increased number of dead pups, lower number of pups at birth and pups still alive at PPD 4. These effects are considered to be secondary effects of systemic toxicity apparent at this dose level. Effects were found to be reversible within recovery period. The NOAEL was determined to be 300 mg/kg bw/d.

 

Phenylethyl Isobutyrate was tested for its mutagenic potential in a bacterial reverse mutation assay in concentrations of 50 to 5000 µg per plate and in a second experiment with 5 to 1500 µg per plate, in the presence and absence of a metabolic activation system (rat liver S9-mix), respectively. In the absence of S9-mix the substance was found to be bacteriotoxic towards the strain TA 102 at 500 µg per plate and towards the strains TA 1535, TA 1537, TA 98 and TA 100 at 1500 µg per plate. Bacteriotoxicity was not observed at any concentration tested in the presence of S9-mix. No significant increase in the mutation frequency of the tested strains in the absence and presence of a metabolic activation system was noted. Thus, indicating that Phenylethyl Isobutyrate under the experimental conditions is not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

 

Mutagenic potential of the test item towards mammalian cells was studied using Chinese hamster (V79) cells. Two independent experiments were conducted. In the first experiment concentrations of 60.3 to 193.0 µg per mL and 1.9 to 120.7 µg per mL with and without S9 mix, respectively, were tested for 4 hours. In the second experiment concentrations of 7.5 to 300 µg per mL were tested for 24 hours and 30 to 960 µg per mL for 4 h, both without metabolic activation. Cytotoxicity was observed after 4 h treatment at 60.3 µg per mL for both with and without metabolic activation. After 24 h, cytotoxicity was observed at a concentration of 241.3 µg per mL and without application of S9 mix. Treatment with the test item did not increase the mutation frequency in both experiments at any concentration level, either with or without metabolic activation. Therefore, the test item is considered to be non-mutagenic to V79 cells at the HPRT locus.

The test item was also assessed for its potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in vitro in both the absence and presence of metabolic activation by rat liver S9 mix. In a preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. Concentrations chosen for the micronucleus assay ranged from 50 to 400 μg per mL for the non-activated 4 and 24-hour exposure groups, and from 100 to 1000 μg per mL for the S9-activated 4-hour exposure group. Substantial cytotoxicity was observed at dose levels greater than 250 µg per mL in the non-activated 4 and 24-hour exposure groups. No cytotoxicity was observed at any dose level in the S9-activated 4-hour exposure group. Visible precipitate was observed in the treatment medium at dose levels greater than 750 µg per mL in the S9-activated 4-hour exposure group. The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level. Thus, the test item is considered to not induce micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

 

 

Toxicokinetic analysis of Phenylethyl Isobutyrate

Phenylethyl Isobutyrate is a pale yellow liquid at room temperature with a molecular weight of 192.25 g/mol. The substance is moderately soluble in water (160 mg/L at 20 °C). The log Pow of Phenylethyl Isobutyrate was determined to be 3.5. The substance has a low vapour pressure of 45 Pa at 25 °C.

 

Absorption

Generally, oral absorption is favoured for molecular weights below 500 g/mol. Moderate water solubility of 160 mg/L enables the substance to partly dissolve in the gastrointestinal fluids and thus it may be considered that direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across membranes with the bulk passage of water takes place. However, the moderate log Pow value of 3.5, which is favourable for passive diffusion, would be the more important property here regarding absorption. Therefore, it can be concluded that Phenylethyl Isobutyrate likely becomes bioavailable following the oral route, as indicated by its physic chemical properties. This assumption is confirmed by the results of the acute oral toxicity study as well as the combined repeated dose toxicity study (OECD 422). In the acute toxicity study, considerable clinical signs, as well as mortalities were observed following administration (leading to LD50 values of >5000 mg/kg bw).

Due to the low vapour pressure of 45 Pa of Phenylethyl Isobutyrate it is unlikely that the substance will be available as a vapour. However, it this case, absorption via inhalation route would be possible due to the moderate log Pow value, enabling uptake directly across the respiratory tract epithelium by passive diffusion.

Dermal absorption can also take place, favoured by the log Pow value, and also by the small molecular weight of the substance. Indeed, clinical effects and mortality at the limit dose of 5000 mg/kg bw were reported (please refer to IUCLID section 7.2.3).

 

Distribution

As mentioned above, the physicochemical properties of Phenylethyl Isobutyrate make systemic bioavailability very likely following oral, inhalative and dermal uptake.

After being absorbed into the body, Phenylethyl Isobutyrate is likely distributed into cells and intracellular concentrations may be higher than extracellular concentrations due to its slightly lipophilic properties (log Pow 3.5). This applies especially for fatty tissues.

Phenylethyl Isobutyrate is unlikely to have a bio-accumulative potential, because it is not highly lipophilic (log Pow is not greater than 4) and there are no other physic-chemical properties indicating bio-accumulating properties. As a combined repeated dose toxicity study (OECD 422) in rats did not reveal any signs of target organ toxicity or other indications for an accumulation in any organ or tissue, there is also no evidence for an accumulative property of this compound. Furthermore, it is stated in literature that the breakdown products are readily metabolized and excreted (see below).

 

Metabolism

Based on its structure, Phenylethyl Isobutyrate may be considered to possibly undergo hydrolysis in contact with aqueous fluids into the corresponding carboxylic acid (isobutyric acid) and phenylethyl alcohol. This process is also highly likely catalyzed by carboxylesterases, (Mutschler, 2001). Hydroxyl group and carboxylic acid are typical functional groups of substrates for phase II enzymes such as UGTs (UDP-glucuronosyltransferase) and GSTs (glutathione-S-transferases) (Marquat & Schäfer, 2004). Conjugated phenylethyl alcohol with glucuronic acid as well as with glycine was already demonstrated in the urine of rabbits (JECFA, 2003). Isobutyric acid, however, is conjugated with Co-enzyme-A and, following β-oxidation, integration into endogenous citric acid cycle is considered (BG Chemie, 1997). Summarizing, the substance and its hydrolytic cleavage products are considered to be readily metabolized and, with this, detoxified. This consideration is supported by findings of in vitro genotoxicity studies applying a metabolizing system (mammalian S9 mix). In both the bacterial reverse mutation assay and the in vitro micronucleus assay the substance showed no cytotoxicity when incubated together with S9. However, substantial cytotoxicity was observed when the substance was applied without metabolizing system in both assays. This is further supported by an HPRT assay, where cytotoxicity was observed irrespective of S9 application or not.

 

Excretion

Phenylethyl Isobutyrate will not be excreted in its non-hydrolysed form. One degradation product, phenylethyl alcohol, is excreted in the urine as conjugate with glycine or glucuronic acid (JECFA, 2003). The other hydrolysis product, isobutyric acid, is rapidly metabolised, integrated in endogenous pathways and thus in main parts excreted as CO2 in the breath (DiVincenzo & Hamilton, 1979). The assumption of rapid excretion is supported by findings of the acute oral toxicity study as well as the combined repeated dose study (OECD 422). In both studies surviving animals recovered rapidly when treatment was determined.