Fatty acids, C18 unsaturated, ethyl & methyl esters

Endpoint summary

Description of key information

The target substance Fatty acids, C18 unsaturated, ethyl & methyl esters is expected to be readily absorbed through the gastrointestinal tract after oral administration. In contrast, dermal and inhalation absorption are considered to be low. The esters will be hydrolysed in the gastrointestinal tract to the respective fatty acid moieties (mainly C18:1, C18:2 or alpha C18:2) and methanol or ethanol which facilitates absorption. The alcohol components methanol and ethanol can dissolve into GI fluids. Thus, these alcohols will be readily absorbed through the GI tract. The highly lipophilic fatty acids are absorbed by micellar solubilisation. Within the epithelial cells, fatty acids are (re)-esterified with glycerol to triglycerides. Methanol or ethanol and the respective fatty acids will be distributed within the organism. The major metabolic pathway for branched fatty acids is the beta-oxidation pathway for energy generation. Methanol or ethanol are mainly oxidized to the respective acid and/or glucuronidated. The excretion will mainly be as CO₂ in expired air; with a smaller fraction excreted in the urine. No bioaccumulation will take place, as excess triglycerides are stored and used as the energy need rises. There is also no potential for methanol or ethanol to accumulate in adipose tissue as methanol and ethanol are soluble in water.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Toxicokinetic-Assessment

Basic toxicokinetics

There are no studies available in which the toxicokinetic behaviour of Fatty acids, C18 unsaturated, ethyl & methyl esters has been investigated.

Therefore, in accordance with Annex VIII, Column 1, Section 8.8.1, of Regulation (EC) No 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2014), assessment of the toxicokinetic behaviour of Fatty acids, C18 unsaturated, ethyl & methyl esters is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physico-chemical and toxicological properties according to Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2014) and taking into account further available information on structural analogue substances.

The substance of Fatty acids, C18 unsaturated, ethyl & methyl esters is a multi-constituent substance specified by esters of ethanol and methanol with fatty acids C18 unsaturated (source canola oil) resulting in monoesters which meet the definition of an UVCB substance. The main fatty acid components are oleic acid (C18:1, 61%), linoleic acid (C18:2, 21%) and linolenic acid (C18:3, 9-11%).

Fatty acids, C18 unsaturated, ethyl & methyl esters is an oily liquid at 20°C which has a molecular weight ranging from 284.5 – 312.5 g/mol and a water solubility of ≥ 0.021 mg/L. The calculated log Pow value is ≥ 7.8 (KOWWIN v1.68) (Erler, 2015) and the vapour pressure is calculated to be < 0.001 Pa at 20 °C (SPARC v4.6) (Erler, 2015).

 

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2014).

Oral

In general, molecular weights below 500 and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract by passive diffusion. Lipophilic compounds may be taken up by micellar solubilisation by bile salts, but this mechanism may be of particular importance for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) as these would otherwise be poorly absorbed (Aungst and Shen, 1986; ECHA, 2014).

When assessing the potential of Fatty acids, C18 unsaturated, ethyl & methyl esters to be absorbed in the gastrointestinal (GI) tract, it has to be considered that fatty acid esters will undergo to a high extent hydrolysis by ubiquitous expressed gastrointestinal enzymes (Lehninger, 1993; Mattson and Volpenhein, 1972). Thus, due to the hydrolysis the predictions based upon the physico-chemical characteristics of the intact parent substance alone may no longer apply but also the physico-chemical characteristics of the breakdown products of the ester; the respective alcohol moieties (methanol and ethanol) and the corresponding fatty acids, mainly C18:1, C18:2 and C18:3.

The high log Pow value (≥ 7.8) and the low water solubility (≥ 0.021 mg/L) of the parent compound indicate that absorption may be limited by the inability to dissolve into GI fluids. However, micellular solubilisation by bile salts may enhance absorption, a mechanism which is especially of importance for highly lipophilic substances with log Pow > 4 and low water solubility (Aungst and Shen, 1986). Regarding molecular weight, the breakdown products methanol (32.04 g/mol) or ethanol (46.07 g/mol) and oleic acid (282.47 g/mol), linoleic acid (280.47 g/mol) and linolenic acid (278.55 g/mol), respectively, are generally favourable for absorption. The alcohol components methanol and ethanol are highly water-soluble and have low molecular weights and can therefore dissolve into GI fluids. Thus, these alcohols will be readily absorbed through the GI tract. The highly lipophilic fatty acids are absorbed by micellar solubilisation (Ramirez et al., 2001). Within the epithelial cells, fatty acids are (re)-esterified with glycerol to triglycerides.

Exemplarily, experimental data of Ethyl oleate (CAS 111-62-6), which is, with a percentage of ca. 45%, one of the main components of Fatty acids, C18 unsaturated, ethyl & methyl esters, confirmed this assumption: The absorption, distribution, and excretion of 14C-labelled Ethyl oleate was studied in Sprague Dawley rats after a single, oral dose of 1.7 or 3.4 g/kg bw. It was shown that the test material and/or its breakdown products was absorbed to approximately 70–90% (Bookstaff et al., 2003).

Moreover, studies on acute oral toxicity with Fatty acids, C18 unsaturated, ethyl & methyl esters showed no signs of systemic toxicity resulting in LD50 values > 5000 mg/kg bw in the rat (Wnorowski, 1995; Lowe, 2015). Furthermore, available data on subacute or subchronic oral toxicity of the main component Ethyl oleate (CAS 111-62-6) and the structural analogue Methyl laurate (CAS 111-82-0) showed no adverse systemic effects resulting in NOAELs of ≥ 1000 mg/kg bw/day (Bockstaff, 2004; Tanaka, 2000). The lack of systemic toxicity cannot be equated with a lack of absorption but rather with a low toxic potential of Fatty acids, C18 unsaturated, ethyl & methyl esters and the breakdown products themselves.

Overall, systemic bioavailability of Fatty acids, C18 unsaturated, ethyl & methyl esters and/or the respective cleavage products in humans is considered likely after oral uptake of the substance.

Dermal

There are no data available on dermal absorption of Fatty acids, C18 unsaturated, ethyl & methyl esters. On the basis of the following considerations, the dermal absorption of the substance is considered to be low:

To partition from the stratum corneum into the epidermis, a substance must be sufficiently soluble in water. Thus, with a water solubility ≥ 0.021 mg/L mg/L, dermal uptake of the substance is likely to be low. In addition, for substances having an octanol/water partition coefficient above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and thus limit absorption across the skin. Furthermore, uptake into the stratum corneum itself may be slow.

The dermal permeability coefficient (Kp) can be calculated from log Pow and molecular weight (MW) applying the following equation described in US EPA (2014):

log(Kp) = -2.80 + 0.66 log Pow – 0.0056 MW

The Kp was calculated for the 5 main constituents of the substance (please refer to Table 1). QSAR calculations confirmed this assumption, as low dermal flux rates ranging from 1.08E-4 – 7.91E-5 mg/cm²per h were calculated indicating only a low dermal absorption potential for the components of Fatty acids, C18 unsaturated, ethyl & methyl esters (please refer to Table 1, Dermwin v2.02, EpiSuite 4.1).

Table 1: Dermal absorption values for the main components of Fatty acids, C18 unsaturated, ethyl & methyl esters (calculated with Dermwin v 2.02, Epiweb 4.1)

Component	Structural formula	Flux (mg/cm2/h)
Ethyl oleate	C20 H38 O2	3.77E-5
Ethyl linoleate	C20 H36 O2	4.31E-5
Methyl oleate	C19 H36 O2	7.91E-5
Ethyl-9,12,15-octadecatrienoate	C20H34O2	4.93E-5
Methyl linoleate	C19H34 O2	1.08E-4

 

In addition, available data on acute dermal toxicity of Fatty acids, C18 unsaturated, ethyl & methyl esters, showed no systemic toxicity resulting in LD50 values > 5000 mg/kg bw (Lowe, 2015).

Moreover, a skin irritation study with the main component Methyl linoleate (CAS 112-63-0) showed only mild irritating effects (Pitterman, 1992).

Overall, taking into account the physico-chemical properties of Fatty acids, C18 unsaturated, ethyl & methyl esters, the QSAR calculations and available toxicological data on structural analogue substances, the dermal absorption potential of the substance is anticipated to be low.

Inhalation

Fatty acids, C18 unsaturated, ethyl & methyl esters has a low vapour pressure of < 0.001 Pa at 20 °C thus being of low volatility. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapours, gases, or mists is not significant.

However, the substance may be available for respiratory absorption in the lung after inhalation of aerosols, if the substance is sprayed. In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract (ECHA, 2014). Lipophilic compounds may be taken up by micellular solubilisation but this mechanism may be of particular importance for highly lipophilic compounds (log P >4), particularly those that are poorly soluble in water (1 mg/L or less) that would otherwise be poorly absorbed.

In addition, available data on acute inhalation toxicity of Fatty acids, C18 unsaturated, ethyl & methyl esters showed no systemic toxicity resulting in a LC50 value> 2.02 mg/L (Wnorowski, 2003).

Based on the physical state and the physico-chemical properties of Fatty acids, C18 unsaturated, ethyl & methyl esters, systemic bioavailability in humans is considered likely after inhalation of aerosols with aerodynamic diameters below 15 μm.

Distribution and accumulation

Distribution of a compound within the body depends on the physicochemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than the extracellular concentration particularly in fatty tissues (ECHA, 2014).

As the parent compound Fatty acids, C18 unsaturated, ethyl & methyl esters will be hydrolysed prior to absorption (as discussed above), the distribution of the intact substance is not relevant but rather the distribution of the breakdown products of hydrolysis. The absorbed products of hydrolysis, methanol or ethanol, and the respective fatty acid moieties can be distributed within the body.

The alcohol moieties methanol and ethanol are small, water soluble substances with log Pow values of -0.824 and 1.16, respectively, which will be distributed in aqueous compartments of the organism. The fatty acids (mainly C18:1, C18:2 or C18:3) are also distributed in the organism and can be taken up by different tissues. They can be stored as triglycerides in adipose tissue depots or they can be incorporated into cell membranes (Masoro, 1977). Substances with high water solubility like methanol or ethanol do not have the potential to accumulate in adipose tissue due to their low log Pow values.

Overall, the available information indicates that the cleavage products, methanol or ethanol, and the respective fatty acids will be distributed within the organism.

Highly lipophilic substances tend in general to concentrate in adipose tissue, and depending on the conditions of exposure may accumulate. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, it is generally the case that substances with high log Pow values have long biological half-lives. The high log Pow of > 5 implies that Fatty acids, C18 unsaturated, ethyl & methyl esters may have the potential to accumulate in adipose tissue (ECHA, 2014). However, as further described in the section metabolism below, esters of alcohols and fatty acids undergo esterase-catalysed hydrolysis, leading to the cleavage products methanol or ethanol and the respective fatty acids. Therefore, the intact parent compound is not assumed to accumulate as hydrolysis takes place before absorption and distribution.

The first cleavage products, methanol or ethanol, are soluble in water (log Pow values of -0.824 and 1.16, respectively), (HSDB). Consequently, there is no potential for methanol or ethanol to accumulate in adipose tissue. The second cleavage product, mainly C18:1, C18:2 or C18:3 fatty acids, can be stored as triglycerides in adipose tissue depots or be incorporated into cell membranes.

This is also shown by experimental data available for Ethyl oleate (CAS 111-62-6), which is, with a percentage of ca. 45%, one of the main components of Fatty acids, C18 unsaturated, ethyl & methyl esters. The absorption, distribution, and excretion of¹⁴C labelled Ethyl oleate was studied in Sprague Dawley rats after a single, oral dose of 1.7 or 3.4 g/kg bw (Bookstaff, 2003). At sacrifice (72 h post-dose), mesenteric fat was the tissue with the highest concentration of radioactivity. The other organs and tissues had very low concentrations of test material-derived radioactivity.

At the same time, fatty acids are also required as a source of energy. Thus, stored fatty acids underlie a continuous turnover as they are permanently metabolized and excreted. Bioaccumulation of fatty acids only takes place, if their intake exceeds the caloric requirements of the organism.

In summary, the available information on Fatty acids, C18 unsaturated, ethyl & methyl esters indicates that no significant bioaccumulation of the parent substance in adipose tissue is expected. The breakdown products of hydrolysis, methanol or ethanol, and the respective fatty acids will be distributed within the organism. Bioaccumulation of the breakdown products is considered to be limited (pleased refer to “Metabolism” and “Excretion”).

 

Metabolism

Esters of fatty acids are hydrolysed to the corresponding alcohol (methanol or ethanol) and fatty acid moieties by esterases (Fukami and Yokoi, 2012). Depending on the route of exposure, esterase-catalysed hydrolysis takes place at different places in the organism: After oral ingestion, esters of alcohols and fatty acids undergo enzymatic hydrolysis already in the gastro-intestinal fluids. In contrast, substances which are absorbed through the pulmonary alveolar membrane or through the skin enter the systemic circulation directly before entering the liver where hydrolysis will basically take place.

The first cleavage products, methanol or ethanol, are mainly oxidized to the respective acid and/or are glucuronidated (HSDB, 2012). 

The second cleavage products, mainly C18:1, C18:2 or C18:3 fatty acid, are stepwise degraded by beta-oxidation. Beta-oxidation is an important metabolic pathway for fatty acids for energy generation. In this multi-step process, the fatty acids are at first esterified into acyl-CoA derivatives and subsequently transported into cells and mitochondria by specific transport systems. In the next step, the acyl-CoA derivatives are broken down into acetyl-CoA molecules by sequential removal of 2-carbon units from the aliphatic acyl-CoA molecule. Further oxidation via the citric acid cycle leads to the formation of H₂O and CO₂(Lehninger, 1993; Stryer, 2002). The omega- and alpha-oxidation, alternative pathways for oxidation, can be found in the liver and the brain, respectively (CIR, 1987).

Following absorption into the intestinal lumen, fatty acids are re-esterified with glycerol to triacylglycerides (TAGs) and included into chylomicrons for transportation via the lymphatic system and the blood stream to the liver. In the liver, fatty acids can be metabolised in phase I and II metabolism.

Available genotoxicity data from the structural related analogue substance Methyl laurate (CAS 111-82-0) do not indicate any genotoxic properties. In particular, an Ames-test (National Institute of Health Sciences, 2000), an in vitro chromosomal aberration test (Buskens, 2010) and an in vitro mammalian gene mutation assay (Verspeek-Rip, 2010) were consistently negative. Therefore, no indication of a genotoxic reactivity of structurally related fatty acid esters is indicated.

Excretion

Based on the metabolism described above, Fatty acids, C18 unsaturated, ethyl & methyl esters and its breakdown products will be metabolised in the body to a high extent. For Fatty acids, C18 unsaturated, ethyl & methyl esters, the main route of excretion is expected to be by expired air as CO₂after metabolic degradation. The second route of excretion is expected to be by biliary excretion via the faeces due to low water solubility. For the cleavage products, the main routes are renal excretion via the urine and exhalation as CO₂.

Experimental data of Ethyl oleate (CAS 111-62-6), which is, with a percentage of ca. 45%, one of the main components of Fatty acids, C18 unsaturated, ethyl & methyl esters, are regarded exemplarily. The absorption, distribution, and excretion of¹⁴C labelled Ethyl oleate was studied in Sprague Dawley rats after a single, oral dose of 1.7 or 3.4 g/kg bw (Bookstaff et al., 2003). The main route of excretion of radioactivity in the groups was via expired air as CO₂. Excretion of¹⁴CO₂was rapid in the groups, as 12 h after dosing 40-70% of the administered dose was excreted in expired air (consistent with beta-oxidation of fatty acids). Female rats had a higher percentage of radioactivity expired as CO₂than the corresponding males. A second route of elimination of radioactivity was via the faeces. Faecal elimination of Ethyl oleate appeared to be dose-dependent. At the dose of 1.7 g/kg bw, 7–8% of the administered dose was eliminated in the faeces. At the dose of 3.4 g/kg bw, approximately 20% of the administered dose was excreted in the faeces. Renal elimination was minimal, with approximately 2% of the radioactivity recovered in urine over 72 h post-dose for the groups (Bookstaff et al., 2003).

 

A detailed reference list can be found in the CSR and in section 13 of the dossier.