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Description of key information

Based on physicochemical characteristics, particularly water solubility 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 some effects at very high doses (above 2000 mg/kg bw). Bioaccumulation of ethyl benzoate or its breakdown products ethanol or benzoic acid will not occur. Enzymatic hydrolysis of ethyl benzoate is supposed to be complete. Ethanol is oxidized to acetaldehyde and degraded via the tricarboxylic acid cycle. Benzoic acid is conjugated to hippuric acid and excreted via urine.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Toxicokinetic profile of ethyl benzoate

Acute oral toxicity studies conducted on rabbits and rats revealed an LD50 value of >2000 mg/kg bw. In a publication it was furthermore reported that the acute toxicity after inhalation in rats lies over the saturated vapour concentration of the test substance. In acute dermal toxicity studies conducted on ethyl benzoate itself as well as on read across substances (methyl benzoate, isopropyl benzoate, butyl benzoate), the LD50 values were also found to be greater than 2000 mg/kg bw. Dermal toxicity studies were performed on rabbits, cats, mice and calves.

 

Ethyl benzoate was also concluded not to be irritating to skin or eyes based on test results with the substance itself and the read across substance methyl benzoate. No skin sensitising properties were revealed either with the read across substance methyl benzoate or with the substance ethyl benzoate itself. The studies were conducted on mice, guinea pigs and humans.

 

In a chronic oral repeated dose toxicity study with rats conducted with the free acid of the substance (read across to benzoic acid), no treatment related adverse effects and no signs of toxicity to reproduction were found up to the highest test concentration of 500 mg/kg bw/day. Rats were exposed to 0.5 and 1 % benzoic acid in two groups of 20 male and 20 female rats in a chronic feeding study. 1 % benzoic acid in the food is equivalent to 500 mg/kg bw/day. The negative control group consisted of the same amount of animals and received untreated food. The first two generations were treated lifelong, the 3rd generation was treated for 16 weeks and the 4th generation until breeding. No effects were observed in any of the generations except for a liver weight increase in the females of the 3rd generation, but this increase was not considered to be caused by the treatment. Therefore, the NOAEL was determined to be 500 mg/kg bw/day.

 

Ethyl benzoate was tested for its mutagenic potential in a bacterial reverse mutation assay in concentrations of 15 to 5000 µg per plate in the presence and of 5 to 5000 µg per plate in the absence of a metabolic activation system (S9-mix). In the absence of S9-mix the substance was found to be bacteriotoxic towards the strain TA102 at 500μg/plate, towards the strain TA98 at 1500μg/plate, and towards the strains TA100, TA1535, and TA1537 at 5000μg/plate. In the presence of S9-mix ethyl benzoate was bacteriotoxic towards the strain TA102 at 1500 µg/plate and towards the strains TA98, TA100, and TA1535 at 5000 µg/plate. The test compound failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. In conclusion, these results indicate that ethyl benzoate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence andabsence of a metabolizing system.

 The read across substance methyl benzoate was tested for its mutagenic properties in mammalian cells.Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. As a conclusion, the test item was shown to be non-mutagenic to V79 cells at the HPRT locus. The test item methyl benzoate (read across), dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item were close to the range of the solvent control values and within the range of the laboratory historical control data. In conclusion, it can be stated that under the experimental conditions reported, the test item methyl benzoate did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required concentration.

Toxicokinetic analysis of ethyl benzoate

Ethyl benzoate is a colourless liquid at room temperature with a molecular weight of 150.1745 g/mol. The substance is soluble in water (720 mg/L at 25 °C). The log Pow of ethyl benzoate was determined to be 2.59. Ethyl benzoate has a low vapour pressure of 24 Pa at 20 °C.

 

Absorption

Generally, oral absorption is favoured for molecular weights below 500 g/mol. Furthermore, the relatively high water solubility of 720 mg/L enables the substance to readily dissolve in the gastrointestinal fluids, allowing direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across membranes with the bulk passage of water. The moderate log Pow value of ethyl benzoate is favourable for passive diffusion. Taken together, the physiochemical properties indicate that ethyl benzoate becomes bioavailable following the oral route. This assumption is confirmed by the results of the acute toxicity studies. These results did not lead to classification of the substance, but at least mortality was observed (leading to LD50 values of >2000 mg/kg bw).

 

Due to the low vapour pressure of ethyl benzoate it is unlikely that the substance will be available as a vapour, but if it is the case absorption via inhalation route is possible due to the water solubility and the moderate log Pow value, enabling uptake directly across the respiratory tract epithelium by passive diffusion.

 

Dermal absorption will also take place, favoured by the water solubility and the log Pow value, and also by the size of the molecule. Indeed, some clinical effects (diarrhoe in rabbits, salivation, tremor, muscular incoordination at a very high dose of 10000 mg/kg bw in cats) and mortality at doses over 2000 mg/kg bw in rabbits were reported (please refer to IUCLID section 7.2.3).

 

Distribution

As mentioned above, the physicochemical properties of ethyl benzoate favour systemic absorption following oral, inhalative and dermal uptake.

After being absorbed into the body, ethyl benzoate is most likely distributed into the interior part of cells due to its slightly lipophilic properties (log Pow 2.59) and in turn the intracellular concentration may be higher than extracellular concentration particularly in adipose tissues.

Ethyl benzoate does not have an accumulative potential, as it is reported that benzoate esters are in general rapidly hydrolysed by carboxylesterases into benzoic acid and aliphatic alcohols.The enzyme mediated hydrolysis occurs in the blood, other body fluids and most body tissues, but predominantly carboxylesterases are found in hepatocytes (JECFA, 2002). As a chronic feeding study in rats conducted on benzoic acid 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. Besides that, it is stated in literature that the breakdown products are further metabolized and excreted (see below).

 

Metabolism

  As mentioned above, benzoate esters are generally hydrolysed by carboxylesterases into benzoic acid and aliphatic alcohols. This enzymatic reaction follows a first order kinetic. Ethyl benzoate was shown to have the longest half-life time of all alkyl benzoate esters. The experimentally determined half-life time for ethyl benzoate was 3.5 h (Nielsen & Bundgaard, 1987).Metabolism of ethyl benzoate is considered to be complete, and the metabolites are not supposed to be more toxic than the parent compound. This assumption is supported by the results obtained in thein vitrogenetic toxicity tests conducted on ethyl benzoate and methyl benzoate in the presence of metabolic activation.

 

After hydrolysis of ethyl benzoate the resulting aliphatic alcohol (ethanol) is oxidized to a more polar metabolite (acetaldehyde) and further metabolized in the tricarboxylic acid cycle (JECFA, 2002). Benzoic acid is undergoing phase II reactions, and, after mainly being conjugated to hippuric acid, rapidly excreted via urine (IPCS & IOMC, 2000; Abdo et al., 1985).

 

Excretion

Ethyl benzoate will not be excreted in its unhydrolized form. The first degradation product, ethanol, and its metabolism and excretion are well known.

The second hydrolysis product, benzoic acid, is metabolised by conjugation to hippuric acid and renally excreted.

 

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