(Z)-N-octadec-9-enylhexadecan-1-amide

Endpoint summary

Additional information

Toxicokinetics, metabolism and distribution

The substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is a secondary fatty amide formed from long chain saturated fatty acids (C16) with the primary amine oleylamine.

It is a bead-like solid at 20 °C (Croda, 2011) with a molecular weight of 505.90 g/mol. The substance has a melting point of 65 °C at normal pressure (Croda, 2011), water solubility of < 0.01 mg/L at 20 °C (Henkel, 2013), log Pow > 5.7 at 23 °C (Henkel, 2013) and vapour pressure of < 8.3E-05 Pa at 20 °C (Henkel, 2012).

Absorption:

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters to provide information on this potential are the molecular weight, octanol/water coefficient (log Pow) value and water solubility (ECHA, 2017). The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

Oral

The molecular weight of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is higher than 500 g/mol, indicating that the substance is poorly available for absorption (ECHA, 2017). 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 hardly soluble in water (≤ 1 mg/L), which would otherwise be poorly absorbed (ECHA, 2017). The high log Pow in combination with the very low water solubility suggests that any absorption of the substance will likely happen via micellar solubilisation by bile salts (ECHA, 2017).

The absorption potential of a substance may also be derived from oral toxicity data, in which e.g. treatment-related systemic toxicity were observed (ECHA, 2017).

The available acute oral toxicity data on the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) showed an oral LD50 value > 2400 mg/kg bw based on the lack of mortality and systemic effects in rats (Notox C.V., 1986). Moreover, data on the oral repeated dose toxicity in rat is available for the substance, indicating that continuous dietary application of the substance for 90 days did not result in any treatment-related adverse effects up to and including the highest dose level of 1000 mg/kg bw/day (IBR, 1988). Furthermore, no adverse effects were observed in rats after receiving the substance via oral gavage for 5 consecutive days at the relatively high dose level of approx. 5000 mg/kg bw/day (Leberco Laboratories, 1962).

Overall, the available data indicate that substance has a low potential for toxicity via the oral route, although no assumptions can be made regarding the actual amount absorbed based on these experimental data.

The potential of a substance to be absorbed in the (GI) tract may be influenced by chemical changes taking place in GI fluids as a result of pH-dependent hydrolysis, metabolism by GI flora, or by enzymes released into the GI tract. These changes will alter the physicochemical characteristics of the substance and hence predictions based upon the physico-chemical characteristics of the parent substance may no longer apply (ECHA, 2017).

Possible metabolites following hydrolysis of the substance were predicted using the OECD QSAR Toolbox version 3.0 (OECD, 2012). The simulation of acidic and basic hydrolysis of the substance resulted in the formation of two metabolites, identified to be the corresponding saturated long-chain fatty acid stearic acid (C16) and the primary amine oleylamine after hydrolysis of the parent compound.

However, having regard to the in vivo situation, acidic hydrolysis of the substance in the stomach is not expected to occur, since it shows a very low solubility in water. This assumption is further supported by hydrolysis data on the structurally related water-insoluble long-chain fatty acid amide oleamide, showing a negligible rate of hydrolysis after incubation for 4 h at 37 °C in simulated gastric fluid containing the hydrolase pepsin (Cooper et al., 1995). In contrast, simulated intestinal fluid enriched with a mixture of several digestive enzymes (pancreatin) and bile salts was found to significantly increase the rate of hydrolysis of oleamide to about 95% after incubation for 4 h at 37 °C, suggesting that the environmental conditions in the intestinal fluid in vivo may likewise favour hydrolysis of water-insoluble fatty acid amides. However, only in the presence of bile salts a complete hydrolysis of the fatty acid amide oleamide in intestinal fluid was achieved, indicating that spontaneous micelle formation by the involvement of bile salts seems to be an important prerequisite for the hydrolysis of long-chain fatty acid amides.

In contrast to the predicted acid- or base-catalysed chemical hydrolysis, data from naturally occurring long-chain fatty acid amides suggest that the substance may rather be cleaved via enzymatic action of intestinal hydrolases after uptake into the body.

There is evidence provided from the physiologically occurring, bioactive substances oleamide and anandamide (arachidonylethanolamide) to show that primary and secondary amides derived from long-chain fatty acids are substrates for fatty acid amide hydrolase (FAAH), a serine hydrolase enzyme widely distributed in the body, including small intestine, liver, kidney and brain (Bisogno et al., 2002; Boger et al., 2000; Wei, et al. 2006). In humans, the liver is one of the organs with the highest FAAH expression and, in contrast to rat and mice, both forms of fatty acid amide hydrolase (FAAH-1 and FAAH-2) are expressed here (Wei et al., 2006). Therefore, the final and complete hydrolysis of those minor amounts of fatty acid amides, which might have escaped hydrolysis in the lumen and the cells of the small intestine so far, may be hydrolysed by FAAH enzymes located in the liver.

Data on the in vitro hydrolysis of the substance in freshly prepared rat liver homogenate, which is known to contain typical mammalian amidases such as FAAH, is available (FDRL, 1960). In this study, 3.6 mg of the substance were incubated for 4 h with rat liver homogenate. At the end of the incubation time, total ammonia liberated from the hydrolysis of the parent substance was determined using the aeration technique of Van Slyke and Cullen and corrected for the amount of naturally occurring ammonia in the liver using blank values from the untreated liver homogenates. The results showed that 3.6 mg of the test substance was hydrolysed by ca. 21% within 4 h in the rat liver homogenate, which is considerably low compared to the degree of hydrolysis reported for other secondary fatty amides (Bisogno et al., 2002; Boger et al., 2000).

In summary, for the in vivo situation, it cannot be directly ruled out if the parent substance or a fraction of it may be absorbed unchanged by micellar solubilisation and be hydrolysed within the body. Therefore, in a worst case approach, the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is anticipated to be enzymatically hydrolysed to the long-chain saturated fatty acidstearic acid (C16)as well as the primary amine oleyl amine.

In general, free fatty acids are readily absorbed by the intestinal mucosa after hydrolysis from triglycerides. Within the epithelial cells, fatty acids are (re-)esterified with glycerol to triglycerides. In general, short-chain or unsaturated fatty acids are more readily absorbed than long-chain, saturated fatty acids (Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1962, 1964).

The second hydrolysis product, oleyl amine, is anticipated to be oxidatively deaminated by monoaminooxidases to yield the corresponding aldehyde and ammonia (Hayes, 2001).

Ammonia, which is liberated from the hydrolysis product oleylamine, is an endogenously occurring molecule resulting from various metabolic processes, including the catabolism of amino acids, amines, nucleic acids, glutamine and glutamate in peripheral tissues (especially in muscle, liver and kidney). Most of the naturally occurring ammonia (ca. 0.23 mol/day) is formed in the gastrointestinal tract, especially in the colon, by hydrolysis of dietary proteins. In the intestine, ammonia is also produced from glutamine or by the rehydrolysation from urea to ammonium (Kuntz and Kuntz, 2008). Ammonia is freely diffusible and toxic to the mammalian organism. However, under physiological conditions, more than 90% of ammonia resulting from metabolic degradation is available as non-diffusible ammonium, resulting in cellular accumulation (Kuntz and Kuntz, 2008; Lehninger, 1993). In the gastrointestinal system, ammonia is readily absorbed in the portal circulation and to a great part detoxified in liver via the urea cycle (Kuntz and Kuntz, 2008; Lehninger, 1993).

In conclusion, based on the available information, the physicochemical properties and molecular weight of the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) suggest poor oral absorption. However, the substance is anticipated to undergo enzymatic hydrolysis in the gastrointestinal tract and absorption of the hydrolysis products may also be relevant.

Dermal

In general, the physical state may already be taken into consideration for a crude estimation of the absorption potential of a substance, which means that dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Furthermore, the dermal uptake of substances with a high water solubility of > 10 g/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal), in particular if water solubility is high. In contrast, log Pow values < –1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low (ECHA, 2017).

The (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is a solid with very low water solubility, thus indicating a poor dermal absorption potential (ECHA, 2017). The high log Pow suggests that the rate of skin penetration by the substance may be limited by the rate of transfer between the stratum corneum and the epidermis (ECHA, 2017).

Apart from the physico-chemical properties, further criteria may apply to assume the dermal absorption potential of the substance.

In general, substances that show skin irritating or corrosive properties may enhance penetration by causing damage to the surface of the skin. Furthermore, if a substance has been identified as a skin sensitiser, then some uptake must have occurred although it may only have been a small fraction of the applied dose (ECHA, 2017).

The experimental animal data on the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) show that no significant skin irritation and no signs of systemic intoxication occurred, which excludes enhanced penetration of the substance due to local skin damage (Notox C.V., 1986b). Furthermore, no skin reactions attributable to a sensitisation reaction and no systemic effects were observed in the skin sensitisation study with the substance (Notox C.V., 1986).

Furthermore, data on dermal toxicity may indicate whether a substance may be absorbed, if signs of systemic toxicity were clearly attributable to treatment (ECHA, 2017).

Consistent with the data on skin irritation and sensitisation, there is no indication for clinical signs of toxicity and any other treatment-related adverse effects from the acute dermal toxicity study with the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6), resulting in an dermal LD50 > 2000 mg/kg bw in rat (Harlan Laboratories Ltd, 2012). Thus, consistent with the data from acute oral toxicity, a low potential for acute dermal toxicity has been demonstrated, although no information on the actual amount of absorbed substance may be derived from these observations.

Overall, based on the available information on physicochemical properties, the dermal absorption potential of the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is predicted to be low.

Inhalation

As the vapour pressure of the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is very low (8.3E-05 Pa at 20 °C), the volatility is also low. Therefore, the potential for exposure to vapours and subsequent absorption via inhalation during normal use and handling is considered to be negligible.

In general, particles with an aerodynamic diameter < 100 μm have the potential to be inhaled, whereas only particles with an aerodynamic diameter < 50 μm can reach the thoracic region and those < 15 μm may enter the alveolar region of the respiratory tract (ECHA, 2017). Data on the particle size distribution of the substance demonstrate that the inhalable fraction of the substance is considerably low, as it contains only 0.03% of particles with an aerodynamic diameter < 500 µm (Croda, 2011). Therefore, under normal conditions of handling, human exposure to the substance via the inhalation route is negligible. Moreover, if any inhalation exposure may occur, the molecular weight, log Pow and water solubility of the substance are suggestive of very low absorption across the respiratory tract epithelium, preferably by micellar solubilisation.

Hydrolases present in the lung lining fluid may also hydrolyse the substance, hence making the hydrolysis products of the substance, the primary fatty amine oleylamine and the corresponding long-chain fatty acids (C16), available for inhalative absorption.

However, due to the information available (low volatility and no inhalable particle size fraction), absorption via inhalation route is assumed to be unlikely, but in case exposure via inhalation should actually occur, absorption is expected to be identical compared to the oral route which is considered to be sufficiently conservative for hazard assessment.

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 extracellular concentration, particularly in fatty tissues (ECHA, 2017).

Considering the worst case situation, the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) will mainly be absorbed in the form of the hydrolysis products. Therefore, the primary fatty amine oleylamine and the long-chain fatty acid (C16) are the most relevant components to assess for the substance.

After being absorbed, fatty acids are (re-)esterified along with other fatty acids into triglycerides and released into chylomicrons, which are transported in the lymph to the thoracic duct and eventually to the venous system. Upon contact with the capillaries, enzymatic hydrolysis of chylomicron triacylglycerol fatty acids by lipoprotein lipase takes place. Most of the resulting fatty acids are taken up by adipose tissue and re-esterified into triglycerides for storage. Triacylglycerol fatty acids are likewise taken up by muscle tissue and oxidised in order to generate energy, or they are released into the systemic circulation and transported in chylomicrons or lipoproteins and returned to the liver (IOM, 2005; Johnson, 1990; Lehninger, 1993; Stryer, 1996).

There is strong evidence that the primary amine oleyl amine will be readily distributed within the organism as experimental animal data on several alkyl amines, e.g. octanamine, demonstrated that alkyl amines are rapidly distributed to the lung, brain, heart, spleen, kidneys and liver (Committee for Risk Assessment, 2011).

Taken together, the potential hydrolysis products of the substance are anticipated to distribute systemically in the organism.

Metabolism:

The potential metabolism of the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) initially occurs via hydrolysis of the amide bond resulting in saturated and mono-unsaturated long-chain fatty acids (C16 and C18:1) and ammonia. Besides chemical hydrolysis, fatty acid amides may be cleaved via enzymatic action of hydrolases, e.g. FAAH, present in the GI tract and other compartments of the body, e.g. the liver. In contrast, substances which are absorbed through the pulmonary alveolar membrane or through the skin may enter the systemic circulation directly before entering the liver where hydrolysis is likely to take place (ECHA, 2017).

A major metabolic pathway for linear fatty acids is the beta-oxidation which is one of the main mechanisms required 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. The complete oxidation of mono-unsaturated fatty acids such as oleic acid, requires an additional isomerisation step. Further oxidation via the citric acid cycle leads to the formation of H₂O and CO₂ (Lehninger, 1993). Alternative pathways for long-chain fatty acids include the omega-oxidation at high concentrations (WHO, 1999). The fatty acids can also be conjugated (by e.g. glucuronides, sulphates) to form more polar products that are easily excreted in the urine.

The primary amine oleyl amine may be oxidatively deaminated by monoaminooxidases to yield the corresponding aldehyde and ammonia (Hayes, 2001). The aldehyde may be further oxidised via the enzymatic action of aldehyde dehydrogenase to the corresponding carboxylic acid, which may be fed into further metabolic pathways such as beta-oxidation (Lehninger, 1993).

Ammonia, resulting from the deamination of the hydrolysis product oleylamine, may be transported to the liver, where it will be converted to urea via the urea cycle. About two-thirds of the ammonia transported to the liver is detoxified via the urea cycle in the periportal hepatocytes, whereas the remaining part is trapped by periportal hepatocytes involved in the glutamine cycle (Kuntz and Kuntz, 2008; Lehninger, 1993). The urea formed in periportal hepatocytes diffuses into the blood and is then transported to the kidneys for re-absorption or final excretion (Lehninger, 1993). However, urea transported in the blood stream may also be taken up into the lumen of the gastro-intestinal tract, in a process termed ‘urea nitrogen salvaging’, where bacterial ureases can cleave urea to provide nitrogen for the synthesis of amino acids and peptides, which may also be reabsorbed by the host mammalian circulation (Stewart and Smith, 2005). Glutamine, the non-toxic transport form of ammonia, is generated in periportal hepatocytes via the action of the enzyme glutamine synthetase (Kuntz and Kuntz, 2008). Hepatic glutamine may be released into blood, distributed to other tissues and fed into the synthesis of amino acids (Lehninger, 1993).

The potential metabolites following enzymatic metabolism of the substance were predicted using the OECD QSAR Toolbox version 3.0 (OECD, 2012). This QSAR tool predicts which metabolites may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the gastrointestinal tract. Thirty-nine hepatic metabolites and 8 dermal metabolites were predicted for the substance. The amide bond is cleaved in both the liver and skin, and the hydrolysis products (the long-chain saturated fatty acids (C16) as well as the primary fatty amine oleylamine) may be further metabolised. Besides hydrolysis, liver and skin metabolites of the substance are mainly the product of beta-oxidation of the C16 fatty acid and C18:1 fatty acid, the latter resulting from the oxidative deamination of oleyl amine and subsequent aldehyde dehydrogenase-dependent oxidation of the corresponding aldehyde. The metabolites formed in the skin are expected to enter the blood circulation and have the same fate as the hepatic metabolites. Up to 140 metabolites were predicted to result from all kinds of microbiological metabolism after hydrolysis of the substance.

Furthermore, the available data from the substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) provide evidence that the substance is not activated to reactive metabolites in the presence of an artificial metabolic system in vitro, since studies performed on genotoxicity (Ames test, gene mutation in mammalian cells in vitro, chromosome aberration assay in mammalian cells in vitro) consistently showed negative results independent of metabolic activation (Jones, 1990; Debets and Enninga, 1986; Linscombe, 1991a; Linscombe, 1991b).

Excretion:

The saturated long-chain fatty acids resulting from hydrolysis of the substance and the monounsaturated fatty acid resulting from the metabolism of the hydrolysis product oleylamine will be further metabolised in order to generate energy or stored as lipids in adipose tissue or used for further physiological functions, e.g. incorporation into cell membranes (Lehninger, 1993). Therefore, the fatty acid metabolites are not expected to be excreted to a significant degree via the urine or faeces, but they are expected to be excreted via exhaled air as CO₂ or stored as described above.

Most of the urea resulting from the detoxification of ammonia in the liver will be transported to the kidneys, where it will either be re-absorbed or directly passed into the urine (Lehninger, 1993).

Taken together, the available data support the assumption that the major portion of the substance may be cleaved after absorption, and the resulting hydrolysis products may either be utilised in physiological pathways or may be excreted from the organism.

A detailed reference list is provided within the CSR.